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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.
Fibronectin fragments cause release and degradation of collagen-binding molecules from equine explant cultures. Previous experiments have shown that addition of fragmented fibronectin can induce cartilage chondrolysis. In this study we investigated the fate of the collagen- and cell-binding molecules Cartilage oligomeric matrix protein (COMP) and chondroadherin. Methods: Equine articular cartilage explants were stimulated with the C-terminal and the N-terminal heparin-binding fragments of fibronectin respectively, and the conditioned media were analysed by both quantitative (ELISA) and qualitative (mass spectrometry, Western blots) methods. Results: Both COMP and chondroadherin were released in a dose-d...
Nucleotide sequence of equine erythropoietin and characterization of region-specific antibodies. To determine the full-length complementary DNA (cDNA) sequence of equine erythropoietin (EPO) and to develop region-specific antibodies to differentiate equine EPO (eEPO) and human EPO (hEPO). Methods: RNA and lysate extracted from renal tissues of an adult Thoroughbred. Methods: Full-length cDNA was determined by use of a reverse transcriptase-polymerase chain reaction assay and a rapid amplification of cDNA ends method. The deduced amino acid sequence was compared with sequences of EPO reported for other species. Furthermore, 4 synthetic peptides were designed in 2 distinctive parts of the e...
Human chorionic gonadotropin-dependent regulation of 17beta-hydroxysteroid dehydrogenase type 4 in preovulatory follicles and its potential role in follicular luteinization. 17Beta-hydroxysteroid dehydrogenase type 4 (17betaHSD4) has a unique multidomain structure, with one domain involved in 17beta-estradiol inactivation. The objective of the study was to investigate the regulation of 17betaHSD4 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The equine 17betaHSD4 cDNA was cloned and was shown to encode a 735-amino acid protein that is highly conserved (81-87% identity) compared with other mammalian orthologs. RT-PCR/Southern blot analyses were performed to study the regulation of 17betaHSD4 transcripts in equine preovulatory follicles ...
Interaction of fibronectin type II proteins with membranes: the stallion seminal plasma protein SP-1/2. Seminal plasma of mammalians contains, among others, proteins that are characterized by the fibronectin (Fn) type II module. Our knowledge about the structure and the physiological function of seminal Fn type II proteins mainly originates from studies on PDC-109, the bovine representative of this protein family. The present work focuses on the equine protein SP-1/2 (also named HSP-1/2) with particular emphasis on its interaction with lipid membranes by employing the intrinsic protein fluorescence and a number of spin-labeled and fluorescent lipid analogues. The results indicate that the intera...
Generation and characterisation of an equine macrophage cell line (e-CAS cells) derived from equine bone marrow cells. Macrophages play a pivotal role in the pathophysiology of many diseases by mediating the host immune response to infections and intoxications. The species-specific activation of macrophages and the differential response in cytokine production impedes the extrapolation of results between species. Therefore, the aim of this study was to isolate and immortalise macrophages from equine bone marrow (BM) cells in order to study equine-specific signalling pathways. The isolated BM-derived macrophages (referred to as e-CAS cells) showed proliferation kinetics similar to that of standardised cell lines...
Blastocyst formation rates in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. This study was conducted to evaluate in vivo and in vitro development of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. Oocytes were collected from slaughterhouse-derived ovaries, matured in vitro, and injected with frozen-thawed stallion sperm. In vivo development was assessed after transfer of injected oocytes to the oviducts of recipient mares. Mares were killed 7.5-8.5 days after transfer and the uterus and oviducts flushed for embryo recovery. Of 132 injected oocytes transferred, 69 (52%) were recovered; of these, 25 (36%) were blastocysts with a blastocoe...
An in vitro biomechanical comparison of a prototype intramedullary pin-plate with a dynamic compression plate for equine metacarpophalangeal arthrodesis. To compare the biomechanical properties of a prototype intramedullary pin-plate (IMPP) implant specifically designed for equine metacarpophalangeal (MCP) arthrodesis with a dynamic compression plate (DCP) system. Methods: In vitro biomechanical testing of paired cadaveric equine forelimbs with a simulated traumatic disruption of the suspensory apparatus, stabilized by one of two methods for MCP arthrodesis. Methods: Twenty-one pairs of adult equine cadaveric forelimbs. Methods: Each forelimb had the distal sesamoidean ligaments severed to create a disrupted suspensory apparatus. For each forel...
In vitro inhibition of blood cholinesterase activities from horse, cow, and rat by tetrachlorvinphos. The organophosphorus insecticide tetrachlorvinphos (TCVP) is commonly used as a feed-through larvicide in many livestock species, including cattle and horses. Cholinesterase (ChE) activity in blood (generally plasma or whole blood) is often employed to assess organophosphorus insecticide intoxication in animals as well as humans. In many species, including horse and man, plasma contains predominantly butyrylcholinesterase whereas red blood cells in all species express exclusively acetylcholinesterase. To evalulate the comparative interaction of TCVP with blood ChEs in different species, we com...
Novel classical MHC class I alleles identified in horses by sequencing clones of reverse transcription-PCR products. Improved typing of horse classical MHC class I is required to more accurately define these molecules and to extend the number identified further than current serological assays. Defining classical MHC class I alleleic polymorphism is important in evaluating cytotoxic T lymphocyte (CTL) responses in horses. In this study, horse classical MHC class I genes were analyzed based on reverse transcription (RT)-PCR amplification of sequences encoding the polymorphic peptide binding region and the more conserved alpha 3, transmembrane and cytoplasmic regions followed by cloning and sequencing. Primer s...
Parathyroid hormone (PTH) secretion, PTH mRNA and calcium-sensing receptor mRNA expression in equine parathyroid cells, and effects of interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha on equine parathyroid cell function. Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid gland in response to changes in ionized calcium (Ca(2+)) concentrations. In this study, we measured PTH secretion, and PTH mRNA and calcium-sensing receptor (CaR) mRNA expression by equine parathyroid chief cells in vitro. We also evaluated the effects of interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha on PTH secretion, and PTH and CaR mRNA expression. The relationship between PTH and Ca(2+) was inversely related. PTH secretion decreased from 100% (day 0) to 13% (day 30). PTH mRNA expression decline...
Xenogenous fertilization of equine oocytes following recovery from slaughterhouse ovaries and in vitro maturation. The in vitro production (IVP) of equine embryos using currently available protocols has met limited success; therefore investigations into alternative approaches to IVP are justified. The objective of this study was to evaluate the feasibility of xenogenous fertilization and early embryo development of in vitro matured (IVM) equine oocytes. Follicular aspirations followed by slicing of ovarian tissue were performed on 202 equine ovaries obtained from an abattoir. A total of 667 oocytes (3.3 per ovary) were recovered from 1023 follicles (recovery rate, 65%). Oocytes underwent IVM for 41 +/- 2 h...
Generation and characterization of an EICP0 null mutant of equine herpesvirus 1. The EICP0 gene (gene 63) of equine herpesvirus 1 (EHV-1) encodes an early regulatory protein that is a promiscuous trans-activator of all classes of viral genes. Bacterial artificial chromosome (BAC) technology and RecE/T cloning were employed to delete the EICP0 gene from EHV-1 strain KyA. Polymerase chain reaction, Southern blot analysis, and DNA sequencing confirmed the deletion of the EICP0 gene and its replacement with a kanamycin resistance gene in mutant KyA. Transfection of rabbit kidney cells with the EICP0 mutant genome produced infectious virus, indicating that the EICP0 gene is not...
Evaluation of in vitro properties of di-tri-octahedral smectite on clostridial toxins and growth. Clostridial colitis and endotoxaemia of intestinal origin are significant causes of morbidity and mortality in horses. Intestinal adsorbents are available for treatment of these conditions; however, little information exists supporting their use. Objective: To evaluate the ability of di-tri-octahedral smectite to bind to Clostridium difficile toxins A and B, C. perfringens enterotoxin and endotoxin, inhibit clostridial growth and the actions of metronidazole in vitro. Methods: Clostridium difficile toxins, C. perfringens enterotoxin and endotoxin were mixed with serial dilutions of di-tri-octa...
Biomechanical investigation of the association between suspensory ligament injury and lateral condylar fracture in thoroughbred racehorses. Objective-To determine whether partial transection of the medial branch of the suspensory ligament (MBSL) alters equine third metacarpal bone (MC3) condylar surface strains and forelimb, distal joint angles in a manner consistent with promotion of lateral condylar fracture. Study Design-In vitro biomechanical experiment. Sample Population-Right forelimbs from 7 Thoroughbred horse cadavers. Methods-Lateral and medial MC3 condylar, dorsal and abaxial, bone surface strains and distal joint angles were measured both before and after partial transection of the MBSL during in vitro axial limb compre...
Babesia bovis merozoites invade human, ovine, equine, porcine and caprine erythrocytes by a sialic acid-dependent mechanism followed by developmental arrest after a single round of cell fission. Babesia bovis infections have only been observed in bovine species in contrast to Babesia divergens that also can infect humans, sheep and rodents. Using an in vitro assay that assesses invasion of erythrocytes by free merozoites after a 1-h incubation period, it was shown that specificity is not determined by host-specific interactions associated with invasion. Human erythrocytes were invaded more efficiently than bovine erythrocytes whereas erythrocytes of sheep, pigs and horses were invaded only slightly less efficiently. In contrast, goat erythrocytes were refractory to efficient invasion....
Characterization and comparison of the responses of equine digital arteries and veins to endothelin-1. To compare the responses of equine digital arteries (EDAs) and equine digital veins (EDVs) to endothelin-1 (ET-1) and determine the role of the endothelium and type of receptors involved in the modulation and mediation of those responses, respectively. Methods: 5 to 9 palmar digital vessels/experiment from 28 healthy horses. Methods: Rings of dissected vessels were mounted under tension between force transducer wires in organ baths containing Krebs-Henseleit solution at 30 degrees C. Responses of EDAs and EDVs (with intact [+e] or denuded [-e] endothelium) to cumulative concentrations of ET-1 ...
Effect of platelet-activating factor (PAF) on stallion sperm motility, capacitation and the acrosome reaction. Phospholipids are an essential component of all mammalian cells; platelet activating factor (PAF=1-O-alkyl-acetyl-sn-glycero-3-phosphocholine) is a signalling phospholipid that has many biological properties in addition to platelet activation. PAF receptors have been detected on stallion spermatozoa; therefore, the aim of this study was to evaluate the effect of synthetic PAF on the motility, capacitation and the acrosome reaction of stallion spermatozoa. Treatment of ten stallion semen samples with 10(-4)-10(-13) mol PAF l(-1) resulted in significant differences in motility and capacitation (...
Preparation of equine isolated hepatocytes. In this study a detailed description of the equine hepatocyte isolation procedure is presented. Livers were obtained from horses slaughtered at the local slaughterhouse. For blood removal and liver preservation the following steps are suggested: perfusion with the oxygenated HBSS (0-2 degrees C, with continuous flow of 500-800 ml/min for 3-6 min), protection from ischemia injury by flushing with ice-cold University of Wisconsin Solution (UW, flow rate of 500-800 ml/min), and finally immersion of the liver lobe in UW solution (2 degrees C) during its transport to the laboratory. For equine isol...
Effects of acetylcysteine and migration of resident eosinophils in an in vitro model of mucosal injury and restitution in equine right dorsal colon. To evaluate the in vitro protective effects of acetylcysteine and response of resident mucosal eosinophils in oxidant-induced injury to tissues of right dorsal colon of horses. Methods: 9 adult horses. Methods: Gastrointestinal mucosa was damaged in vitro with 3 mM hypochlorous acid (HOCl), with and without prior exposure to 6mM acetylcysteine. Control tissues were not exposed to HOCl or acetylcysteine. Control and damaged tissues were incubated in Krebs-Ringer-bicarbonate solution and tissue resistance measured during 240 minutes. Tissue permeability to radiolabeled mannitol was also used to ...
Novel purification method for mammalian seminal plasma phospholipid-binding proteins reveals the presence of a novel member of this family of protein in stallion seminal fluid. A family of bull seminal plasma (BSP) phospholipid-binding proteins (BSP proteins), potentiate heparin- and HDL-induced capacitation. The homologous proteins have been purified from stallion and boar seminal plasma, and detected in low concentrations in other mammalian seminal plasma. In this study, we developed a new isolation method for mammalian seminal plasma choline phospholipid-binding proteins wherein they are present in low concentrations. The method is based on the interaction of this family of proteins with egg yolk low-density lipoprotein fraction (LDF). In order to demonstrate the ...
Rhodococcus equi secreted antigens are immunogenic and stimulate a type 1 recall response in the lungs of horses immune to R. equi infection. Rhodococcus equi is an opportunistic pathogen in immunocompromised humans and an important primary pathogen in young horses. Although R. equi infection can produce life-threatening pyogranulomatous pneumonia, most foals develop a protective immune response that lasts throughout life. The antigen targets of this protective response are currently unknown; however, Mycobacterium tuberculosis is a closely related intracellular pathogen and provides a model system. Based on previous studies of M. tuberculosis protective antigens released into culture filtrate supernatant (CFS), a bacterial growth s...
In vitro comparison of equine cancellous bone graft donor sites and tibial periosteum as sources of viable osteoprogenitors. To compare the osteogenic potential of cancellous bone of conventional graft sites with that of one nonconventional site (fourth coccygeal vertebra) and to investigate the tibial periosteum as a donor site with respect to osteogenic potential. Methods: In vitro osteogenic cell culture system. Methods: Eight adult horses. Methods: Cancellous bone or tibial periosteum was aseptically collected and cut into bone chips or periosteal strips of 1 to 2 mm(3) for primary explant cultures. After 2 weeks, primary tissue cultures that yielded a population of osteogenic cells were counted and subcultured ...
A low-level X chromosome mosaicism in mares, detected by chromosome painting. Fluorescence in situ hybridization with the use of the equine X whole chromosome painting probe was carried out on chromosome spreads originating from three mares with poor reproductive performance (infertility, miscarriage or stillbirth). The numbers of analysed spreads were high (105, 300 and 480) and in all three mares a low frequency of mosaicism was identified. The mares had the following karyotypes: 64,XX/63,X/65,XXX (93.6%/5.7%/0.7%), 64,XX/63,X (98.9%/1.1%) and 64,XX/63,X (94.3%/5.7%). The incidence and importance of the low percentage X chromosome mosaicism are discussed.
Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa. In oocytes from all mammalian species studied to date, fertilization by a spermatozoon induces intracellular calcium ([Ca(2+)](i)) oscillations that are crucial for appropriate oocyte activation and embryonic development. Such patterns are species-specific and have not yet been elucidated in horses; it is also not known whether equine oocytes respond with transient [Ca(2+)](i) oscillations when fertilized or treated with parthenogenetic agents. Therefore, the aims of this study were: (i) to characterize the activity of equine sperm extracts microinjected into mouse oocytes; (ii) to ascertain i...
Inhibition of in vitro maturation of equine oocytes by interleukin 1 beta via specific IL-1 receptors. Interleukin 1 beta (IL-1 beta) inhibits the LH-induced resumption of meiosis of equine oocytes in vitro. The present study was performed to clarify this inhibitory effect of IL-1 beta by testing increasing concentrations of IL-1 beta, and by measuring the effect of addition of IL-1 receptor antagonist (IL-1RA) to the culture medium. The effect of IL-1 beta on epidermal growth factor (EGF)-induced resumption of meiosis was also studied. Cumulus-oocyte complexes (COCs) were collected from subordinate follicles on ovaries obtained from an abattoir. In five distinct experiments, COCs were cultured...
Development and testing of a modular strain measurement clip. A novel, multi-use, low-stiffness and low-cost transducer for measuring in vitro strains has been developed and tested. Currently available strain measurement methods are either too expensive, too complicated or too inflexible for multi-use strain measurement. The stainless-steel modular strain measurement clip introduced here was instrumented with four 350 Omega axial strain gauges in a full Wheatstone bridge configuration to take advantage of commonly available strain gauge amplifier equipment. Adjustable extension arms were designed to allow greater application versatility. The clip was cal...
Anti-protozoal efficacy of medicinal herb extracts against Toxoplasma gondii and Neospora caninum. The purpose of this study was to determine whether alcohol extracts of herbs (Sophora flavescens Aiton, Sinomenium acutum (Thunb.) Rehder and E.H. Wilson, Pulsatilla koreana (Yabe ex Nakai) Nakai ex T. Mori, Ulmus macrocarpa Hance and Torilis japonica (Houtt.) DC.) from South Korea, possess in vitro anti-protozoal activity against cultures of Toxoplasma gondii and Neospora caninum. These herbs have been used as human anti-parasitics in Asian countries for many years. Alcohol extracts of these herbs were serially diluted to final concentrations ranging from 625 to 19.5 ng/ml in media and added ...
Characterisation of movement pattern and velocities of stallion spermatozoa depending on donor, season and cryopreservation. The aim of the study was to compare different types of movement pattern and velocities of stallion spermatozoa depending on cryopreservation during breeding and non-breeding season. Ejaculates were collected from four stallions during May (n = 24) and December (n = 24). Parameters of sperm movement were evaluated by computer-aided sperm analysis (CASA) system, and included percentages of motile spermatozoa, different patterns of motility, the velocity, linearity (LIN), amplitude of lateral head displacement (ALH) and beat-cross frequency (BCF). In winter the average percentages of motility wer...
Ovarian function and morphology in the mare after multiple follicular punctures. In the mare, ultrasound-guided transvaginal oocyte recovery and transfer might offer a way to circumvent the demanding procedures of in vitro embryo production. Before clinical application, the possible consequences for subsequent fertility have to be considered. Objective: To examine ovarian function and morphology in mares after repeated follicular punctures. Methods: A total of 14-26 follicular puncture sessions were conducted on each of 4 Norwegian pony mares over a period of 8 years. The ovaries of these mares were recovered by bilateral ovariectomy or at post mortem and subjected to macr...
Differences in polymorphonucleocyte function and local inflammatory response between horses and ponies. Wound healing proceeds faster in ponies than in horses and complications during healing, such as wound infection, occur less frequently in ponies. Earlier studies suggested that this difference might be related to differences in the initial post traumatic inflammatory response. Objective: That polymorphonuclear leucocyte (PMN) function and profiles of humoral factors in local inflammatory processes are different in horses and ponies. Methods: PMNs were isolated from venous blood of horses and ponies. Chemotaxis and reactive oxygen species (ROS) production was determined. Tissue cages were impl...
