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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.
In vitro comparison of transfixation and standard full-limb casts for prevention of displacement of a mid-diaphyseal third metacarpal osteotomy site in horses. To compare transfixation and standard full-limb casts for prevention of in vitro displacement of a mid-diaphyseal third metacarpal osteotomy site in horses. Methods: 6 forelimbs from 6 horses euthanatized for reasons not related to the musculoskeletal system. Methods: A 30 degrees osteotomy was performed in the mid-diaphysis of the third metacarpal bone. Two 4.5-mm cortical bone screws were placed across the osteotomy site to maintain alignment during casting. Two 6.35-mm Steinmann pins were placed from a lateral-to-medial direction in the distal aspect of the radius. A full-limb cast that inc...
Significance of plasmalemma disruption in bovine and equine spermatozoa. We have investigated fresh and cryopreserved bovine and equine spermatozoa using light and transmission soft X-ray microscopy. Spermatozoa were examined, in the presence or absence of semen, after using Percoll gradient centrifugation and re-suspending in medium. X-ray microscopy provided high resolution (30 nm) transmission images of whole cells in solution with high contrast, while retaining the simple preparation techniques used in light microscopy. We demonstrated translucent, membrane-bound vesicles in the acrosomal and midpiece regions that were similar in size and we noted their inciden...
In vitro effects of 5-hydroxytryptamine and cisapride on the circular smooth muscle of the jejunum of horses. To determine effects of cisapride and 5-hydroxytryptamine (5-HT) on the jejunum of horses. Methods: Jejunal muscle strips from 8 horses. Methods: Muscle strips were suspended in isolated muscle baths. Isometric stress responses to 5-HT and cisapride, with and without specific antagonists, were determined. Results: Muscle strips incubated with atropine and tetrodotoxin responded to 5-HT and cisapride with an increase in contractile force. The 5-HT caused a concentration-dependent increase in contractile amplitude, with a maximum response (Emax) of 1,151+/-214 g/cm2 and a molar concentration tha...
The use of pentoxifylline to improve motility of cryopreserved equine spermatozoa. Pentoxifylline was evaluated as a method to increase motility of cryopreserved equine spermatozoa. In a preliminary experiment, pentoxifylline (3.5 mM or 7.0 mM) was added to extended semen that was chilled to 4 degrees C. Motility was evaluated at 8-h intervals for 48 h. The addition of 3.5 or 7.0 mM pentoxifylline appeared to increase the motility of chilled spermatozoa compared to controls. Based on these results, similar concentrations of pentoxifylline were added to semen either before or after cryopreservation. The addition of pentoxifylline (3.5 or 7.0 mM) to semen before cryopreservati...
In vitro mucolytic activity of recombinant human deoxyribonuclease on equine tracheobronchial mucus. The viscosity of the mucus, its DNA concentration and the size range of the DNA were determined on tracheobronchial samples from 11 horses with lower airway diseases before and after incubation with recombinant human deoxyribonuclease (rhDNase). The horses were divided into two groups on the basis of the cytology of the samples: group A (five horses) with more than 60 per cent neutrophils and group B (six horses) with fewer than 50 per cent neutrophils. The mean mucus viscosity and DNA concentration in the preincubation samples were significantly higher in group A than in group B, and there wa...
Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions. The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas ...
vCLAP, a caspase-recruitment domain-containing protein of equine Herpesvirus-2, persistently activates the Ikappa B kinases through oligomerization of IKKgamma. vCLAP, the E10 gene product of equine herpesvirus-2, is a caspase-recruitment domain (CARD)-containing protein that has been shown to induce both apoptosis and NF-kappaB activation in mammalian cells. vCLAP has a cellular counterpart, Bcl10/cCLAP, which is also an activator of apoptosis and NF-kappaB. Recent studies demonstrated that vCLAP activates NF-kappaB through an IkappaB kinase (IKK)-dependent pathway, but the underlying mechanism remains unknown. In this report, we demonstrate that vCLAP associates stably with the IKK complex through direct binding to the C-terminal region of IKKgamma....
Characterization of acid phosphatase activities in the equine pathogen Streptococcus equi. Acid phosphatases hydrolyse phosphomonoesters at acidic pH in a variety of physiological contexts. The recently defined class C family of acid phosphatases includes the 32 kDa LppC lipoprotein of Streptococcus equisimilis. To define further the distribution of acid phosphatases in the genus Streptococcus we have examined the equine pathogens Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus. Whole cell assays indicated that these organisms possess two acid phosphatases with activity optima at pH 5.0 and pH 6.0-6.5 and that only the former of these was, like LppC, resis...
Expression and regulation of transcripts encoding two members of the NR5A nuclear receptor subfamily of orphan nuclear receptors, steroidogenic factor-1 and NR5A2, in equine ovarian cells during the ovulatory process. Steroidogenic factor-1 (SF-1, NR5A1a) is a member of the NR5A nuclear receptor subfamily and has been implicated as a key transcriptional regulator of all ovarian steroidogenic genes in vitro. To establish links between the expression of SF-1 and that of the steroidogenic genes in vivo, the objectives of this study were to clone equine SF-1 and examine the regulation of its messenger RNA (mRNA) in follicular cells during human CG (hCG)-induced ovulation. The equine SF-1 primary transcript was cloned by a combination of RT-PCR techniques. Results showed that the transcript was composed of a 5'-...
The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm ...
Characterization of a coronavirus isolated from a diarrheic foal. A coronavirus was isolated from feces of a diarrheic foal and serially propagated in human rectal adenocarcinoma (HRT-18) cells. Antigenic and genomic characterizations of the virus (isolate NC99) were based on serological comparison with other avian and mammalian coronaviruses and sequence analysis of the nucleocapsid (N) protein gene. Indirect fluorescent-antibody assay procedures and virus neutralization assays demonstrated a close antigenic relationship with bovine coronavirus (BCV) and porcine hemagglutinating encephalomyelitis virus (mammalian group 2 coronaviruses). Using previously des...
The effects of methylprednisolone on normal and monocyte-conditioned medium-treated articular cartilage from dogs and horses. To study in vitro (1) the dose-response relationships between proteoglycan metabolism in normal and corticosteroid-treated articular cartilage; (2) long-term proteoglycan metabolism after treatment of articular cartilage with corticosteroids; and (3) the effect of corticosteroids on proteoglycan metabolism in articular cartilage treated with monocyte-conditioned medium (MCM). Methods: Equine and canine articular cartilage explants were treated with corticosteroids and MCM. Proteoglycan synthesis and degradation were measured by radioactive labeling in short-term culture, and the long-term effe...
Comparison of culture and insemination techniques for equine oocyte transfer. This study was designed to test 3 approaches for insemination and transfer of oocytes to recipient mares. Oocytes were recovered transvaginally from naturally cycling donor mares 24 to 26 h after an intravenous injection of 2500 IU of hCG when follicles reached 35 mm in diameter. Multiple oocytes (1 to 4) were transferred surgically into the oviducts of 4 or 5 recipient mares per group. Three groups of transfers were compared: 1) transfer of oocytes cultured in vitro for 12 to 14 h postcollection with insemination of the recipient 2 h postsurgery; 2) transfer of oocytes into the oviduct within...
Centrifugation and addition of glycerol at 22 degres C instead of 4 degrees C improve post-thaw motility and fertility of stallion spermatozoa. The aims of this study were to evaluate the effects of cooling rate to 4 degrees C and temperature at the time of centrifugation/glycerol-addition (freezing extender: INRA82 + 2% egg yolk + 2.5% glycerol) on postcentrifugation recovery rate, post-thaw motility and per-cycle fertility. When centrifugation/glycerol-addition was performed at 4 degrees C (14 ejaculates), a moderate cooling rate (37 degrees C to 4 degrees C in I h) resulted in higher post-thaw motility (45%) than when using a slow cooling rate (37 degrees C to 4 degrees C in 4 h) (39%; P<0.05). When centrifugation/glycerol-addit...
Endotoxin and dietary amines may increase plasma 5-hydroxytryptamine in the horse. Uptake of 5-hydroxytryptamine (5-HT) into platelets is an important mechanism by which low plasma concentrations are maintained, and platelet activation may therefore result in significant release of this vasoconstrictor. The present study examined the kinetics of active uptake of radiolabelled [3H]5-HT by washed equine platelets in vitro, and investigated the effects on this process of 4 other naturally occurring monoamines which may be released from the caecum in conditions of carbohydrate overload. The release of [3H]5-HT by platelets was also studied, since platelet accumulation and activa...
Acrosomal ultrastructure of stallion spermatozoa cryopreserved with ethylene glycol using two packaging systems. The present experiments aimed to examine the substitution of glycerol (G) by ethylene glycol (E) as a cryoprotective agent for stallion spermatozoa. Two different ethylene glycol concentrations (5% and 10%) and also the association of glycerol (2%) and ethylene glycol (3%) (E/G) were studied (Experiment 1). In Experiment 2, two packing systems (0.5 x 4.0 ml) were evaluated using both cryoprotectors. In both experiments, the sperm membrane integrity after freezing was evaluated using transmission electron microscopy. The mean post-thaw motility was 34.25, 36.5, 29.25 and 34.75% for G5%, E5%, E1...
The effects of glucosamine derivatives on equine articular cartilage degradation in explant culture. To determine whether glucosamine-3-sulfate, glucose-3-sulfate (control) and N-acetyl glucosamine inhibit experimentally induced degradation of equine articular cartilage explants similar to glucosamine HCl. Methods: Articular cartilage was obtained from the antebrachio-carpal and middle joints of horses (2-8 years old) killed for reasons unrelated to lameness. Cartilage discs were harvested from the weight-bearing region of the articular surface and cultured. Media were exchanged daily and the recovered media stored at 4 degrees C. On days 1 and 2 lipopolysaccharide (LPS, 10 microg/ml) was add...
4-nitroimidazole binding to horse metmyoglobin: evidence for preferential anion binding. The ionization of 4-nitroimidazole to 4-nitroimidazolate was investigated as a function of ionic strength. The apparent pKa varies from 8.99 to 9.50 between 0.001 and 1.0 M ionic strength, respectively, at 25 degrees C. The ionic strength dependence of this ionization is anomalous. The binding of 4-nitroimidazole by horse metmyoglobin was studied between pH 5.0 and 11.5 and as a function of ionic strength between 0.01 and 1.0 M. The association rate constant is pH-dependent, varying from 24 M(-1)s(-1) at pH 5 to a maximum value of 280 M(-1)s(-1) at pH 9.5 and then decreasing to 10 M(-1)s(-1) a...
Colonization of the stratified squamous epithelium of the nonsecreting area of horse stomach by lactobacilli. Selective adhesion to only certain epithelia is particularly common among the bacterial members of the indigenous microflora of mammals. We have found that the stratified squamous epithelium of the nonsecreting area of horse stomach is colonized by gram-positive rods. The microscopic features of a dense layer of these bacteria on the epithelium were found to be similar to those reported in mice, rats, and swine. Adhering microorganisms were isolated and identified as Lactobacillus salivarius, L. crispatus, L. reuteri, and L. agilis by DNA-DNA hybridization and 16S rRNA gene sequencing techniqu...
In vitro investigation of the effect of prostaglandins and nonsteroidal anti-inflammatory drugs on contractile activity of the equine smooth muscle of the dorsal colon, ventral colon, and pelvic flexure. To determine the in vitro effect of prostaglandin E2 (PGE2), PGF2alpha, PGI2; and nonsteroidal anti-inflammatory drugs (NSAID; ie, flunixin meglumine, ketoprofen, carprofen, and phenylbutazone) on contractile activity of the equine dorsal colon, ventral colon, and pelvic flexure circular and longitudinal smooth muscle. Methods: 26 healthy horses. Methods: Tissue collected from the ventral colon, dorsal colon, and pelvic flexure was cut into strips and mounted in a tissue bath system where contractile strength was determined. Incremental doses of PGE2, PGF2alpha,, PGI2, flunixin meglumine, carp...
In vitro comparison of metaphyseal and diaphyseal placement of centrally threaded, positive-profile transfixation pins in the equine third metacarpal bone. To evaluate in vitro holding power and associated microstructural and thermal damage from placement of positive-profile transfixation pins in the diaphysis and metaphysis of the equine third metacarpal bone. Methods: Third metacarpal bones from 30 pairs of adult equine cadavers. Methods: Centrally threaded positive-profile transfixation pins were placed in the diaphysis of 1 metacarpal bone and the metaphysis of the opposite metacarpal bone of 15 pairs of bones. Tensile force at failure for axial extraction was measured with a materials testing system. An additional 15 pairs of metacarpal bone...
Evaluation of substance P as a neurotransmitter in equine jejunum. To determine whether substance P (SP) functions as a neurotransmitter in equine jejunum. Methods: Samples of jejunum obtained from horses that did not have lesions in the gastrointestinal tract. Methods: Jejunal smooth muscle strips, oriented in the plane of the circular or longitudinal muscle, were suspended isometrically in muscle baths. Neurotransmitter release was induced by electrical field stimulation (EFS) delivered at 2 intensities (30 and 70 V) and various frequencies on muscle strips that were maintained at low tension or were under contraction. A neurokinin-1 receptor blocker (CP-96...
In vitro comparison of the use of two large-animal, centrally threaded, positive-profile transfixation pin designs in the equine third metacarpal bone. To compare the in vitro holding power and associated microstructural damage of 2 large-animal centrally threaded positive-profile transfixation pins in the diaphysis of the equine third metacarpal bone. Methods: 25 pairs of adult equine cadaver metacarpal bones. Methods: Centrally threaded positive-profile transfixation pins of 2 different designs (ie, self-drilling, self-tapping [SDST] vs nonself-drilling, nonself-tapping [NDNT] transfixation pins) were inserted into the middiaphysis of adult equine metacarpal bones. Temperature of the hardware was measured during each step of insertion with ...
Differential localization of protein kinase C isotypes in equine eosinophils and neutrophils. Phorbol esters, which activate protein kinase C (PKC), stimulate equine eosinophil superoxide production and adherence. After showing that superoxide production could be inhibited by the nonselective PKC inhibitors, staurosporine and bisindolymaleimide I, the PKC isotypes in equine eosinophils were characterized, because evidence suggests that individual isotypes may play distinct roles in regulating eosinophil function. Western blots demonstrated that equine eosinophils expressed PKC alpha, beta, delta, epsilon, iota, and zeta. However, unlike the equine neutrophil, the majority of the PKC wa...
In vitro model of equine muscle regeneration. Equine satellite cells are responsible for muscle healing and regeneration in the mature horse. We describe the in vitro cell culture conditions required for clonal populations of equine satellite cells to undergo both proliferation and differentiation. Our hypothesis is that these in vitro conditions model regeneration of muscle and can be used to evaluate potential therapeutics. In this study, 2 areas of satellite cell response were tested: proliferation of clones induced by growth factors, and fusion induced by culture conditions. Equine satellite cell clones showed differences in their res...
Interactions of CRH, AVP and cortisol in the secretion of ACTH from perifused equine anterior pituitary cells: “permissive” roles for cortisol and CRH. To further elucidate the interaction of CRH, AVP and cortisol in the control of ACTH secretion, we used an in vitro perifusion model with dispersed equine anterior pituitary cells. To approximate the in vivo milieu in the horse, CRH was perifused continuously (at 0, 2 and 20 pmol/L) and 5-min pulses of AVP (0, 1, 3 and 10 nmol/L) were given every 30 min in the presence of 0 or 100 nmol/L cortisol. Total (baseline + incremental) ACTH secretion increased as both the CRH (p<0.001) and the AVP (p<0.001) concentration increased and interaction between CRH and AVP was significant (p=0.042). Co...
Interleukin-1 potentiates basal and AVP-stimulated ACTH secretion in vitro–the role of CRH pre-incubation. The acute-phase cytokine interleukin-1 (IL-1) is known to activate the hypothalamic pituitary adrenal axis, primarily via corticotropin releasing hormone (CRH). The aim of this study was to determine whether IL-1beta could directly stimulate ACTH secretion from perifused equine anterior pituitary cells, and whether CRH pre-incubation affected corticotroph responsiveness. Isolated equine anterior pituitary cells were pre-incubated with media containing 10 nM CRH or vehicle for 20 hours before being loaded onto columns and perifused with 0.02 nM CRH and 100 nM cortisol. Columns were given a 5-mi...
Effects of angiotensin II on the acrosome reaction in equine spermatozoa. Angiotensin II is a hormone with a wide array of physiological effects that exerts its effect via interaction with two major subtypes of receptor. The results of this study show that angiotensin II (from 1 to 100 nmol l(-1)) initiates acrosomal exocytosis in equine spermatozoa that have undergone capacitation in vitro in a TALP-TEST (Tyrode's albumin lactate pyruvate; 188.7 mmol TES l(-1), 84.8 mmol Tris l(-1)) buffer with cAMP. The acrosome reaction and sperm viability were assessed with fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) and Hoechst 33258, respectively. The initia...
Effect of eastern equine encephalomyelitis virus on the survival of Aedes albopictus, Anopheles quadrimaculatus, and Coquillettidia perturbans (Diptera: Culicidae). The effect of eastern equine encephalomyelitis (EEE) virus on the survivorship of Aedes albopictus (Skuse), Anopheles quadrimaculatus Say, and Coquillettidia perturbans (Walker) was determined experimentally. Female mosquitoes were allowed to feed on EEE viremic chicks, and survival rates were compared for infected and uninfected mosquitoes. Additionally, the survival of female Cq. perturbans and An. quadrimaculatus intrathoracically (i.t.) inoculated with EEE was compared with controls receiving diluent inoculations. Infection with EEE significantly reduced survival in Cq. perturbans compared...
