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.
Antigenic relationship between Pythium insidiosum de Cock et al. 1987 and its synonym Pythium destruens Shipton 1987. Antigens and rabbit-antisera from holotypes of Pythium insidiosum and P. destruens were prepared to elucidate their antigenic relationship. The antigens and rabbit-antisera of P. insidiosum as well as P. destruens used as a reference system showed that both shared three precipitin bands in common. The antigen and rabbit-antisera of P. destruens and P. insidiosum used as a reference system against other strains isolated from humans and animals with pythiosis, also showed three precipitin bands in common. When we used sera taken from horses with proven pythiosis against antigens of P. insidiosum...
Changes in quality of stallion spermatozoa during cryopreservation: Plasma membrane integrity and motion characteristics. Better procedures for freezing and thawing equine sperm are needed since variable fertility is obtained when cryopreserved sperm are used. To evaluate current methods of freezing equine sperm, we examined spermatozoal quality by means of two new techniques. These measured the integrity of plasma-acrosomal membranes by immunofluorescent analyses of binding of an antibody specific to the acrosome and evaluated eight parameters of spermatozoal motion using a fully automated computerized system. Five ejaculates from each of eight stallions were processed for freezing in egg yolk-lactose extender w...
A growth-promoting factor for human myeloid leukemia cells from horse serum identified as horse serum transferrin. A growth-promoting factor for human myeloid cells was purified to apparent homogeneity from horse serum by a combination of gel filtration, blue Sepharose affinity chromatography, Mono Q anion-exchange chromatography, Mono P chromatofocusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The growth promoter was an iron-bound, single glycopolypeptide chain with a molecular weight of 84,000, an isoelectric point of 5.4 and an amino terminal sequence of Glu-Gln-Thr-Val-Arg-Trp-Cys-Thr-Val-Ser-Asn-His-Glu-Val-Ser-Lys-. According to the results of the amino acid sequence, iron bindi...
Identification and description of alpha-helical regions in horse muscle acylphosphatase by 1H nuclear magnetic resonance spectroscopy. It has been proposed that combination of intraresidue, sequential and longer range nuclear Overhauser enhancements occurring in 1H nuclear magnetic resonance spectra of protein chains folded in a helix show a regular characteristic pattern. As a test case the spectra of horse muscle acylphosphatase were searched for this pattern together with other typical signs of a helical conformation (i.e. chemical shift, coupling constants and slow 2H-H exchange). Two amino acid sequences complying with these requirements were found. Just a few amino acid spin system assignments were then sufficient to lo...
Crystallization and preliminary X-ray study of horse pancreatic lipase. Horse (Equus caballus) pancreatic lipase (EC 3.1.1.3) has been crystallized using the hanging drop method of vapour diffusion at 20 degrees C. The best crystals were grown from an 8 mg/ml solution in 10 to 20% (w/v) polyethylene glycol 8000, 10 mM-MgCl2, 0.1 M-NaCl, 0.1 M-Mes buffer (pH 5.6). They reach dimensions of 0.8 mm x 0.4 mm x 0.6 mm. X-ray examination of the lipase crystals shows that they are orthorombic with a space group P2(1)2(1)2(1). Their cell dimensions are a = 79.8 A, b = 97.2 A c = 145.3 A. Two molecules per asymmetric unit give a Vm value of 2.82 A3/dalton (56% water content...
The structure and properties of horse muscle acylphosphatase in solution. Mobility of antigenic and active site regions. The solution structure of acylphosphatase determined by proton nuclear magnetic resonance spectroscopy is described. The results allow us to discuss the fold of the protein (101 amino acids), to correlate the exposure and the mobility of the backbone with the antigenicity, and to locate the active site.
Identity of the E-series prostaglandin produced by equine chondrocytes and synovial cells in response to a variety of stimuli. Although an E-series prostaglandin has previously been identified in equine inflammatory exudate, the identity of this eicosanoid as PGE2 has not been confirmed. The objective of this study was the specific characterisation of the prostaglandin produced by equine cells in the presence of an inflammatory stimulus. By using two radioimmunoassays, a relatively non-specific PGE2 assay and a more specific PGE1 assay, it has been possible to identify the E-series prostaglandin produced by equine chondrocytes and synovial cells, in response to a variety of stimuli, as PGE2.
Methylation at the CpG doublet in equine adenovirus genome. Viral DNA obtained from the equine adenovirus propagated in equine transitional cell carcinoma (ETCC) cells and in equine fetal dermis cells were compared by cleaving with isoschizomeric restriction enzymes, HpaII and MspI, and then electrophoresed in 1.4 per cent agarose gels. Differences between the HpaII and MspI cleavage patterns were evident in viral DNA obtained only from the equine adenovirus propagated in ETCC cells, suggesting site specific methylation at CpG sequences.
Comparative microsomal oxidation of febantel and its metabolite fenbendazole in various animal species. A comparison has been made of the in vitro metabolism of febantel (FBT) with that of one of its pharmacologically active metabolites fenbendazole (FBZ) using microsomal preparations from liver of sheep, calf, horse, pig, rat, chicken and trout. The oxidation of FBT to the corresponding sulphoxide appeared to be far more rapid with the exception of the trout, than a similar reaction with FBZ. Indeed FBT was further metabolized in several species by cyclization and further oxidation. This observation could have toxicological significance in view of the greater tetratogenic effects of the metabol...
Application of an affinity electrophoretic and in situ oxidation method to the study of the equine protease inhibitory proteins. An affinity method was developed to investigate the interaction between protease and protease inhibitor by incorporating a protease incubation step into a two-dimensional electrophoretic separation of the plasma protease inhibitory proteins. This involved the application of the isoelectric focusing gel to filter paper saturated in the protease of choice before being placed on the second-dimensional polyacrylamide electrophoresis gel. General protein staining or immunoblotting was used to detect the protein or ligand in the complex. An in situ oxidation method was developed using the reagent ch...
Pharmacological characterization of mare uterus motility with special reference to calcium antagonists and beta-2-adrenergic stimulants. 1. Uterine motility was studied in vitro in the myometrial tissue obtained from pregnant and non-pregnant mares. 2. The spontaneous contractions of the preparations were not modified by tetrodotoxin, by anticholinergics, antiadrenergics, histamine H1 and H2 blockers, antiserotoninergic and opioid antagonists; but disappeared in Ca2+ and Na+ free medium. 3. beta 2-adrenergic stimulants like salbutamol and hexoprenaline and the calcium channel blockers nifedipine and verapamil were effective inhibitors of the amplitude of phasic contractions (ID50S for salbutamol and nifedipine were 7.7 nM and 1...
Purification of F(ab’)2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with high neutralization activity, purity and yield. Pooled horse plasma containing antibodies against Crotalus durissus terrificus whole venom were digested with pepsin at an enzyme-substrate ratio of 8:1, pH 3.1, for 40 min and the F(ab')2M fragments purified by adding 8.7% caprylic acid (pH 5.0). For comparison, F(ab')2B purified by precipitation with ammonium sulphate and uncleaved IgG purified with caprylic acid were also prepared. Fab' fragments were obtained by reduction and alkylation of F(ab')2B. The anti-whole C.d. terrificus venom titers, determined by Dot-Blot were 12,800 (IgG), 6400 [F(ab')2B], 4800 [F(ab')2M] and 3200 (Fab'B). Immu...
The continuum of events leading to maternal recognition of pregnancy in mares. Endometria from pregnant mares are able to produce PGF in vitro, but when co-incubated with conceptus membranes the amount and rate of PGF production is considerably reduced. To estimate the molecular weight of conceptus factors that inhibited PGF production, Day-14 conceptus membranes were placed inside bags constructed of dialysis tubing and co-incubated with endometria from Day-14 pregnant mares. PGF production was significantly reduced when membranes were in bags with molecular weight exclusion limits of 12,000, 6000, and 3500, but not of 1000, suggesting that conceptus PGF-inhibitory fact...
Comparative effects of cholera toxin, Salmonella typhimurium culture lysate, and viable Salmonella typhimurium in isolated colon segments in ponies. Isolated segments of left dorsal colon and a side-to-side colocolostomy (between the left ventral colon and left dorsal colon) were surgically created in 6 adult ponies. Four segments, each separated by an empty segment, were inoculated (20 ml) with 1 of the following 4 solutions: phosphate buffered saline solution (PBSS)/1% polyethylene glycol (PEG); purified cholera toxin in PBSS/1% PEG (5 micrograms cholera toxin/ml of PBSS/1% PEG); lyophilized Salmonella typhimurium UCD 1755 culture lysate, reconstituted in PBSS/1% PEG; and viable S typhimurium UCD 1755 (10(8) organisms/ml of PBSS/1% PEG)....
Kinetic and inhibitory characteristics of serum angiotensin-converting enzyme from nine mammalian species. 1. Serum angiotensin-converting enzyme activities were obtained from nine mammalian species: rat, mouse, horse, sheep, guinea pig, hamster, rabbit, dog and man. 2. Kinetic constants (Km and Vmax) using hippuryl-L-histidyl-L-leucine as substrate and inhibitory constants (I50 and Ki) for captopril were determined for the serum ACE of each species. 3. There were important differences in the kinetic and inhibitory constants (Kms went from 6.6 mM to 1.21 mM for hamster and guinea pig; I50 ranged from 2100 nM to 3 nM for mouse and sheep) as well as differences in enzyme activity of the different spe...
Enzymatic deacylations of esterified saccharides–III. Comparison of de-esterifications by serum esterases from different sources. 1. 14C-labelled methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside (1) was used as a substrate for esterases from rabbit, guinea pig, mouse, donkey, pig, horse, sheep and human sera. 2. Stepwise de-esterification of the diester substrate 1 occurred with rabbit, guinea pig and mouse serum. Data on time-course experiments and kinetic data are reported. 3. The use of donkey, pig, horse, sheep and human serum led to the migration of the 2-O-pivaloyl group in substrate 1 to the position 4- in the sugar molecule, followed by stepwise de-esterifications of both 1 and the newly formed methyl 4,6-di-O-pi...
Preliminary investigations on the effects of a Strongylus vulgaris larval extract, mononuclear factors and platelet factors on equine smooth muscle cells in vitro. Factors involved in the proliferation of equine vascular smooth muscle cells were studied in vitro. The most prominent proliferative responses in cultured vascular smooth muscle cells were induced by Strongylus vulgaris larval antigen extract (LAE) and platelet-derived factors. Less significant proliferative responses were obtained with conditioned media from S. vulgaris LAE stimulated and from unstimulated equine mononuclear leukocytes. Additionally, vascular smooth muscle cells exposed to S. vulgaris LAE developed numerous perinuclear vacuoles and were more spindle-shaped than control or smo...
Lipopigments in veterinary pathology: pathogenesis and terminology. The lipopigments are a heterogenous group of pigments whose pathogenesis and terminology is confused. Whereas there is epidemiological and observational evidence that ceroid is derived from degeneration and peroxidation of unsaturated lipid, the assumption that all so-called lipopigments are similarly formed, is questioned. In particular, recent studies have distanced the pathogenesis of the pigment found in the ceroid-lipofuscinoses from that perceived for ceroid. The importance of protein rather than lipid in the pathogenesis of the pigment of ceroid-lipofuscinosis and of age pigment from th...
Membrane viscosity of lymphocytes and influence of phytohemagglutinin. The membrane viscosity of peripheral blood lymphocytes (PBLs) of equine, bovine and canine was measured by the use of time-resolved fluorescence depolarization technique with 1, 6-diphenyl-1,3,5-hexatriene (DPH). The viscosity values were 0.55, 0.59 and 0.50 poise for equine, bovine and canine PBLs, respectively. These values were compared with steady-state anisotropies and order parameters measured from electron spin resonance (ESR) of 5-doxyl stearic acid. Both values were increased with increase of viscosity. The fluid property of the membranes stimulated with phytohemagglutinin-P (PHA) was...
Biochemical evidence that equine leucocyte antigens W13, W22 and W23 are present on horse major histocompatibility complex class II molecules. A number of horse alloantisera were characterized biochemically as being directed against MHC class I or class II antigens by immunoprecipitation of the corresponding antigens from lysates of biosynthetically radioactively labelled lymphocytes and determination of their molecular weights by SDS-PAGE and fluorography. Sera recognizing A2 and A3 specificities precipitated antigens of 44,000 Daltons molecular weight (class I heavy chain), whereas sera with specificities W13, W22 and W23 precipitated antigens corresponding to class II dimers (30,000 and 32,000 Daltons). Comparison with antigens pr...
In vitro susceptibility of Pseudomonas mallei to antimicrobial agents. Pseudomonas mallei was isolated from pus samples obtained from 34 mallein-positive horses. The isolates were subjected to in vitro sensitivity test using 16 different antimicrobial discs. All isolates (34) were sensitive to sulfamethizole, gentamycin, tetracycline, sulfathiazole, kanamycin, tobramycin, streptomycin and a combination of trimethoprim and sulfamethoxazole while none of them were sensitive to cephalothin, colistin, ampicillin, penicillin and nitrofurantoin. Rifapicin, chloramphenicol and carbenicillin were effective against 32, 26 and 18 isolates respectively. The minimum inhibito...
Studies on the effect of short term, high dose praziquantel treatment against protoscoleces of ovine and equine Echinococcus granulosus within the cyst, and in vitro. Protoscoleces of Echinococcus granulosus were subjected to high concentrations of praziquantel (10-1000 micrograms/ml medium or cyst fluid) for a short, 10 min duration in vitro or in situ within cysts. Drug efficacy was then monitored either by in vitro culture in drug-free medium and/or by passage into rodents. The in vitro test of effectiveness suggested that ovine-derived protoscoleces were more sensitive than those of equine origin, and that the in vitro method of treatment was more successful than that performed in situ. However, the subsequent passage of treated protoscoleces, whether i...
In vivo activation of equine eosinophils and neutrophils by experimental Strongylus vulgaris infections. Eosinophils and neutrophils from ponies with Strongylus vulgaris-induced eosinophilia (eosinophilic ponies; activated eosinophils and neutrophils) were assayed in vitro for chemotactic and chemokinetic responses to zymosan-activated serum (ZAS) using the filter system in Boyden chambers, for Fc and complement (C) receptors using the EA and EAC-rosette assays, respectively, and for phagocytic and bactericidal activities using opsonized Escherichia coli and the acridine orange method. The responses of activated eosinophils and neutrophils in the above assays were compared with those of eosinophi...
Effect of phenytoin on the clinical signs and in vitro muscle twitch characteristics in horses with chronic intermittent rhabdomyolysis and myotonia. In vitro twitch characteristics of the semimembranosus muscle were evaluated in 9 clinically normal horses, in 15 horses with chronic intermittent rhabdomyolysis (CIR) and in 2 horses with myotonia. Effects of phenytoin on in vitro muscle twitch and clinical signs of CIR and myotonia were evaluated in these same horses. Times to 90% relaxation were prolonged in the horses with CIR (mean +/- SEM, 186 +/- 5.9 ms) and in 2 horses with myotonia (197 and 177 ms) compared with those in clinically normal horses (mean +/- SEM, 146 +/- 2.1 ms). Horses with CIR also had significantly (P less than 0.05) ...
Prokinetic effects of cisapride, naloxone and parasympathetic stimulation at the equine ileo-caeco-colonic junction. The electromyogram of the terminal ileum, the caecum and the proximal right ventral colon was recorded in fasted conscious ponies receiving intravenously equiactive doses of pilocarpine (0.05 mg/kg) and carbachol (0.01 mg/kg) as acetylcholine analogues; cisapride (0.1 mg/kg) and metoclopramide (2 mg/kg) facilitating acetylcholine release from myenteric neurones and naloxone (0.05 mg/kg) as an antagonist of the endogenous inhibitory opioid system. Both cisapride and naloxone induced typical migrating spike bursts in the colon associated with contractions of caecal body and caecal base. Both pil...
Pharmacokinetics, bioavailability, and in vitro antibacterial activity of rifampin in the horse. The pharmacokinetics and bioavailability of rifampin were determined after IV (10 mg/kg of body weight) and intragastric (20 mg/kg of body weight) administration to 6 healthy, adult horses. After IV administration, the disposition kinetics of rifampin were best described by a 2-compartment open model. A rapid distribution phase was followed by a slower elimination phase, with a half-life (t1/2[beta]) of 7.27 +/- 1.11 hours. The mean body clearance was 1.49 +/- 0.41 ml/min.kg, and the mean volume of distribution was 932 +/- 292 ml/kg, indicating that rifampin was widely distributed in the body....
Monoclonal antibody recognizes a conformational epitope in a random coil protein. The antigenic determinants for two monoclonal antibodies directed against horse apo-cytochrome c, a protein of disordered structure, as judged by spectroscopic and hydrodynamic criteria, have been studied by a combination of methods: antigen competition in solution by radio immunoassay and enzyme-linked immunoassay, and differential acetylation of free and antibody-bound antigen. In the latter method the accessibility of lysine residues of the antigen in the antigen-antibody complex is compared to the accessibility in the free antigen. The two antibodies against the heme-free protein do not re...
Opsonins in uterine washings influencing in vitro activity of equine neutrophils. Uterine washings were found to promote neutrophil mediated killing of Streptococcus zooepidemicus. Depletion of complement and/or specific antibody from the washings significantly reduced bactericidal activity. Phagocytosis of yeast by uterine washings was complement dependent. Inhibition of the classical pathway significantly reduced opsonic activity indicating that, in addition to direct activation via the alternate pathway, antibody may also be involved in yeast phagocytosis.
Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion sperm. Transmission electron microscopy was used to confirm that a monoclonal antibody (F79.3E2; class IgG1 kappa) was specifically localized to an antigen in the acrosomal ground substance of stallion sperm. This antibody was used to develop and validate an indirect immunofluorescent procedure to evaluate integrity of the plasma-acrosomal membranes of stallion sperm. The concept was that primary monoclonal antibody would be "shielded" from its acrosomal antigen by an intact plasma membrane. Conversely, sperm with damaged plasma-acrosomal membranes would exhibit green acrosomal fluorescence when view...
Endotoxin-induced procoagulant activity in equine peripheral blood monocytes. Increasing evidence has demonstrated the importance of monocyte procoagulant activity (PCA) in the pathogenesis of coagulopathies in a variety of diseases. Because endotoxin precipitated coagulopathies are common sequelae to intestinal ischemia/endotoxemia in the equine species, we investigated the ability of equine peripheral blood monocytes to express PCA. Monocytes isolated from five healthy adult horses were incubated in vitro with Escherichia coli endotoxin (10 micrograms), and the PCA was measured by the ability of cellular lysates to accelerate the clotting times of equine plasma in a m...