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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 effects of erythromycin, lidocaine, and metoclopramide on smooth muscle from the pyloric antrum, proximal portion of the duodenum, and middle portion of the jejunum of horses. To evaluate effects of erythromycin, lidocaine, and metoclopramide on smooth muscle of the pyloric antrum (PA), proximal portion of the duodenum (PD), and middle portion of the jejunum (MJ) of horses. Sample Population-Strips of smooth muscle from 7 horses. Methods: Isolated muscle strips were suspended in a bath and attached to isometric force transducers. Once stable spontaneous contractions were observed, agents were added. Isometric stress responses were compared with the amplitude of spontaneous contractions. Results: A single dose of erythromycin to the PA increased contractile amplitude...
Consideration of the role of antigenic keratan sulphate reacting to a 1/14/16H9 antibody as a molecular marker to monitor cartilage metabolism in horses. The role of keratan sulphate (KS) as a marker of cartilage metabolism was evaluated by using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n=3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor (IGF)-Ialpha or interleukin (IL)-1alpha for 2 weeks. The sulfated glycosaminoglycans (GAG) and antigenic KS concentrations in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition ELISA using a 1/14/...
Actions and interactions of ADP, 5- HT, histamine and PAF on equine platelets. Platelets are thought to play a role in equine diseases such as acute laminitis and verminous arteritis and may be involved in allergic disease. Mediators implicated in the pathophysiology of these conditions activate platelets and responses may be enhanced by interactions between mediators. The present study compared platelet aggregation, thromboxane production and release of radiolabelled [(3)H]5- HT in response to 5- HT, histamine, ADP and PAF alone and in combination in vitro.PAF caused concentration-related aggregation, [(3)H]5- HT release and thromboxane production. In contrast, ADP caus...
Equine osteoclast-like cells generated in vitro demonstrate similar characteristics to directly isolated mature osteoclasts. We report on novel methods to isolate osteoclasts (OC s) and generate osteoclast-like cells (OCL s) from the bone and bone marrow of the equine femur. OC s were successfully isolated from bone scrapings taken from the endosteal surface of the femurs of three horses. OCL s were generated from bone marrow cells taken from the same animals. The validity of using the formation of OCL s as a method for studying OC differentiation and activity was confirmed by the similar characteristics of these two cells. In particular, they both were multinuclear, expressed the enzyme tartrate resistant acid phos...
Equine immunity to bacteria. The remarkable ability of the horse and other animals to prevent infection by most bacterial pathogens encountered is the result of a complex set of distinct but overlapping defense mechanisms. This article summarizes the current state of knowledge on innate and adaptive immunity to bacterial pathogens and reviews various ways in which some bacteria have evolved in order to evade components of the host response.
The effect of oxytocin on contractility of the equine oesophagus: a potential treatment for oesophageal obstruction. This study was performed to determine the effect of administration of i.v. oxytocin on the contractility of the musculature associated with the equine oesophagus. Nine clinically normal horses were fitted with a nasogastric tube modified with inflatable latex cuffs. These cuffs were connected to piezoelectric pressure recording devices. Oxytocin in 3 different doses or saline controls were administered i.v. in a randomised block pattern. Systolic blood pressure, ECG, heart rate and nasogastric tube cuff pressures were then measured for 60 min. Administration of oxytocin i.v. at 0.11 and 0.22 i...
Spontaneous production of nitric oxide (NO), prostaglandin (PGE2) and neutral metalloproteinases (NMPs) in media of explant cultures of equine synovial membrane and articular cartilage from normal and osteoarthritic joints. Nitric oxide (NO), prostaglandin E2 (PGE2), and the activity of neutral metalloproteinases (NMPs) were measured in conditioned media of equine synovial membrane and articular cartilage explant cultures from horses with normal joints (n = 7) and from horses affected with moderate (n = 7) or severe osteoarthritis (n = 14) as judged by macroscopic appearance. Normal articular cartilage appeared glossy and bluish-white, was of normal thickness and showed no evidence of discolouration, fibrillation or other cartilage discontinuity. Slight discolouration and fibrillation or minor clefts of the carti...
Melanin affinity: a possible explanation of isoxsuprine retention in the horse. Isoxsuprine is used in veterinary medicine as a vasodilating agent. The drug has been detected in the urine of horses up to 6 weeks after the cessation of administration. In the present study, the distribution pattern of 3H-isoxsuprine was investigated using whole body autoradiography in mice to find a possible site of retention. Melanin was the only place of retention identified. Additional in vitro studies showed an affinity of isoxsuprine to both melanin and keratin. The K(d) values were 0.02 mmol/l and 1 mmol/l, and the B(max) values were 0.2 micromol/mg and 2 micromol/mg, respectively. A ...
Affinity of isoxsuprine for adrenoreceptors in equine digital artery and implications for vasodilatory action. We used isolated equine digital arteries to study the vasodilatory mechanism of isoxsuprine, and fowl caecum preparations to investigate the affinity of the drug for beta-adrenoceptors. Isoxsuprine is a potent vasodilator of arterial smooth muscle that has been precontracted by an alpha-adrenoceptor agonist such as noradrenaline (log EC50 = -6.33 [-5.98; -6.68]). The present study indicates that its effect is due to alpha-adrenoceptor blockade since: (1) after a long lasting exposure to cumulative doses of isoxsuprine the vasoconstricting action of noradrenaline cannot be restored; (2) isoxsup...
Proton electron nuclear double resonance from nitrosyl horse heart myoglobin: the role of His-E7 and Val-E11. Electron nuclear double resonance (ENDOR) spectroscopy has been used to study protons in nitrosyl horse heart myoglobin (MbNO). (1)H ENDOR spectra were recorded for different settings of the magnetic field. Detailed analysis of the ENDOR powder spectra, using computer simulation, based on the "orientation-selection" principle, leads to the identification of the available protons in the heme pocket. We observe hyperfine interactions of the N(HisF8)-Fe(2+)-N(NO) complex with five protons in axial and with eight protons in the rhombic symmetry along different orientations, including those of the ...
The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion. The relationship between fertility and susceptibility of sperm DNA to denaturation was determined in a group of 84 actively breeding, clinically fertile stallions. Susceptibility of DNA to denaturation was determined using the sperm chromatin structure assay (SCSA). The SCSA measures, mean of alpha-t (mean alpha t), standard deviation of alpha-t (SD alpha t), and the COMP of alpha-t (cells outside the main population)] were significantly correlated with the percentage seasonal pregnancy rate (SPR; mean alpha t, r = -0.24, P < or = 0.05; % COMP alpha t, r = -0.27, P < or = 0.05); percenta...
Effectiveness of two systems for transporting equine semen. The storage and transport of cooled, liquid semen is an effective way of facilitating the use of desirable stallions for breeding mares located on distant farms. The Equitainer System is the most widely used transport container and it has been shown that it is possible to ship semen in this container and obtain good conception rates. However, the cost of Equitainers is high, and stud-farms that ship large quantities of semen have tended to rely on cheaper alternatives, even though little documentation exists concerning their reliability, especially under extreme temperature conditions. Two dif...
Effects of glutamine, proline, histidine and betaine on post-thaw motility of stallion spermatozoa. The supplementation of the freezing diluent with 3 amino acids (glutamine, proline and histidine) and 1 amino acid-related compound (betaine) in preserving stallion spermatozoa diluted in INRA82 extender containing 2.5% (v/v) glycerol and 2% (v/v) egg yolk (control extender) during freezing and thawing was studied at 0, 40, 80, 120 and 160 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 1). Glutamine and proline were studied at 0, 10, 20, 30, 40, 50, 60, 70 and 80 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 2). In each experiment, spermatozoa were eval...
Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility. The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known ...
Generation of an equine oviductal epithelial cell line for the study of sperm-oviduct interactions. Equine oviductal epithelial cells (OEC) were transformed with simian virus 40 large T antigen (SV 40 T-ag) to create a cell line for the study of the interaction of equine spermatozoa with oviductal epithelium. One cell line was established based on the expression of the SV 40 T-ag and extended lifespan in culture. Immortalized equine OEC retained the characteristics of differentiated OEC such as the formation of monolayers with characteristic epithelial morphology and cell polarization as well as expression of cytokeratin and equine major histocompatibility complex I. Monolayers of immortaliz...
Effect of time during transport of excised mare ovaries on oocyte recovery rate and quality after in vitro maturation. In the mare only a limited number of oocytes can be successfully collected in vivo, so that when large numbers of oocytes are needed for experimentation, ovaries harvested from slaughtered mares must be used. The resulting temperature changes and time intervals mandated by handling and transport of ovaries from the slaughterhouse to the laboratory adversely affect the rate of oocyte recovery and their quality after IVF and maturation. We chose to study the effect of temperature and time in transit of excised ovaries by evaluating rate of oocyte recovery, nuclear maturation stage reached before...
Heme orientation affects holo-myoglobin folding and unfolding kinetics. Native myoglobin (Mb) consists of two populations which differ in the orientation of the heme by 180 degrees rotation (as verified by nuclear magnetic resonance) but have identical absorption spectra and equilibrium-thermodynamic stability. Here, we report that these two fractions of native oxidized Mb (from horse) both unfold and refold (chemical denaturant, pH 7, 20 degrees C) in two parallel kinetic reactions with rate constants differing 10-fold. In accord, the oxidized heme remains coordinated to unfolded horse Mb in up to 4 M guanidine hydrochloride (pH 7, 20 degrees C).
Interaction of bull, stallion and boar seminal plasma proteins and sperms with acidic polysaccharides. The interaction of seminal plasma proteins, sperms and detergent-released sperm proteins of three species with different types of acidic polysaccharides was studied. Heparin-binding activity of boar, bull and stallion seminal plasma proteins, sperms and sperm proteins was compared with their ability to interact with polysaccharides differing in the presence of the sulfate groups or in their saccharide moiety (chondroitin sulfate, dextran sulfate, fucoidan, hyaluronic acid). Bull seminal plasma proteins were characterized by higher affinity to heparin, fucoidan and dextran sulfate, while signif...
Effect of mare’s age and recovery methods on the recovery rate of equine follicular oocytes for IVM procedures. Mares (n = 39) were classified according to age as young (less than 1.5 yr, n = 17) or old (more than 1.5 yr, n = 22) and sacrificed. Ovaries were measured and weighed, and the number of follicles and CL were counted. Follicle size and distribution were recorded (external: > 20 mm, 5 mm, < 5 mm). External follicles were aspirated while internal follicles were sliced. The number and Type of oocytes recovered using each method were recorded. Oocyte recovery rates (oocytes/ovary) resulted in a mean of 0.92 oocytes by aspiration and 1.36 oocytes by additional slicing. The mean numbers of av...
Effect of passive immunization against inhibin on FSH secretion, folliculogenesis and ovulation rate during the follicular phase of the estrous cycle in mares. Physiological roles of inhibin in mares were investigated by means of passive immunization using an antiserum to inhibin that had been raised in a castrated goat. Eight mares were given an intravenous injection of either 100 mL (n = 4) or 200 mL (n = 4) of inhibin antiserum 4 d after a single intramuscular injection of PGF2 alpha on Day 8 after ovulation, 4 control mares were treated with 100 mL castrated goat serum in the same manner. Jugular vein blood samples were collected after treatment with the serum until 192 h post treatment. Follicular growth and ovulations were monitored by ultrasou...
Production of embryos by assisted reproduction in the horse. In vitro embryo production is not yet successful in the horse, largely due to low rates of fertilization in vitro. However, methods to produce embryos from isolated oocytes have been developed. Oocytes may be recovered from living mares by aspiration of the dominant preovulatory follicle by trans-abdominal puncture, and from both preovulatory and immature follicles by trans-vaginal ultrasound-guided puncture. Transfer of in vivo-matured oocytes to the oviducts of bred recipient mares has resulted in good pregnancy rates (75-85%). Little work has been done on transfer of horse oocytes matured i...
Equine retained placenta: technique for and tolerance to umbilical artery injections of collagenase. Under laboratory conditions and in clinical experiments, bacterial collagenase has proven to be effective in hydrolyzing placenta and detaching cotyledon from caruncle in the bovine species. Laboratory studies in which placental samples were incubated with collagenase have also demonstrated that collagenase is 3.7 times more effective in hydrolyzing equine placenta than bovine placenta. This led to the hypothesis that collagenase may be a potential treatment for mares with retained placenta. However, that collagenase may hydrolyze the uterine wall and perforate the uterus was a concern. It was...
Current methods for stallion semen cryopreservation: a survey. Various factors affect the success of AI with frozen-thawed semen in horses. Stallion variability is thought to be one of the major factors, but semen processing and evaluation techniques, thawing protocols, packaging systems and timing of insemination are far from standardized among laboratories. Our objective was to survey current methods for stallion semen cryopreservation used commercially around the world. From the answers to the questions in the survey, we attempted to provide an overview of procedures that are standard as well as those that are used by only few laboratories and to revie...
Bacteriology of preserved stallion semen and antibiotics in semen extenders. Three experiments were conducted to evaluate the effects of different antibiotics in a milk-glucose semen extender on motility of equine sperm and elimination of bacteria following storage of extended semen in vitro. In Experiment 1, 7 antibiotics were compared: amikacin, gentamicin, streptomycin, potassium penicillin, sodium penicillin, ticarcillin, and polymixin B. In Experiment 2, 3 antibiotic treatments were compared: potassium penicillin G, amikacin, or a combination of potassium penicillin G and amikacin. In Experiment 3, 3 antibiotic treatments were compared: potassium penicillin G-amik...
Epididymal maturation affects calcium regulation in equine spermatozoa exposed to heparin and glucose. Spermatozoal function is affected by the ability to regulate intracellular calcium concentrations ([Ca2+]i), and may be influenced by epididymal maturation as well as environmental components. Regulation of [Ca2+]i in ejaculated and epididymal stallion spermatozoa was monitored over time in various media. Spermatozoa from each of 5 pony stallions (3 ejaculate samples and 1 caput and cauda sample) were labeled with the fluorescent calcium indicator probe Indo-1 in a calcium-free modified Tyrode's buffer. Fluorescent emissions were monitored by a dual wavelength spectrofluorometer over 5 h. Calc...
Synteny and regional marker order assignment of 26 type I and microsatellite markers to the horse X- and Y-chromosomes. The hypothesis that the conservation of sex-chromosome-linked genes among placental mammals could be extended to the horse genome was tested using the UCDavis horse-mouse somatic cell hybrid (SCH) panel. By exploiting the fluorescence in-situ hybridization (FISH) technique to localize an anchor locus, X-inactivation-specific transcript (XIST) on the horse X chromosome, together with the fragmentation and translocation of the X- and Y-chromosome fragments in a somatic cell hybrid panel, we regionally assigned 13 type I and 13 type II (microsatellite) markers to the horse X- and Y-chromosomes. T...
A biomechanical comparison of double-plate and Y-plate fixation for comminuted equine second phalangeal fractures. To compare the biomechanical properties, in full limb preparations, of intact second phalanx and a simulated comminuted second phalangeal fracture stabilized with either two bone plates or a custom Y-plate. Methods: In vitro biomechanical assessment of intact limbs and of paired limbs with a simulated second phalangeal fracture stabilized by one of two fixation methods. Animal Population-Thirteen pairs of equine cadaveric forelimbs. Methods: A comminuted second phalangeal fracture was created in six paired cadaveric limbs. For each limb pair, the fracture was stabilized with two plates in one ...
In vitro cyclic biomechanical properties of an interlocking equine tibial nail. To determine cyclic biomechanical properties of gap osteotomized adult equine tibiae stabilized with an equine interlocking nail (EIN). Methods: In vitro experimental biomechanical investigation. Methods: Thirteen adult equine cadaveric tibiae. Methods: Adult equine tibiae with transverse, midshaft, 1-cm gap osteotomies, stabilized with an equine interlocking nail, underwent cyclic biomechanical testing in vitro under axial compression, 4-point bending, and torsion. Different specimens were subjected to different load levels that represented estimated in vivo loads at 2 Hz for 740,000 cycles. ...
Hypoosmotic test in equine spermatozoa. The aim of the study was to evaluate equine sperm membrane integrity using the hypoosmotic swelling (HOS) test and to correlate this test with different sperm parameters in raw and frozen thawed semen. The HOS solutions were made with fructose, sucrose, lactose and sodium citrate each at 300, 150, 100, 50 and 25 mosm. Maximum numbers of swollen spermatozoa were observed in solutions of fructose, sucrose and lactose each at 100, 50 and 25 mosm. Correlations between progressive motility, morphologically normal spermatozoa and the HOS test were r = 0.75 and r = 0.51 in raw semen and r = 0.26 and ...
Quality of stallion semen obtained by a new semen collection phantom (Equidame) versus a Missouri artificial vagina. A study was performed to test a new semen collection device (Equidame phantom) that fractionates the ejaculate by comparing the quality of semen obtained by the Equidame phantom with that obtained by a Missouri artificial vagina. Semen from 4 Finnhorse stallions was collected 4 times per stallion by both methods. Half of the ejaculate was frozen and the other half extended and loaded into 2 Equitainer transport containers (24- and 48-h samples). Motility parameters were determined by a Hamilton-Thorn motility analyzer after cooled storage for 24 and 48 h and again after freezing/thawing. Raw a...
