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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.
Equine catechol estrogen 4-hydroxyequilenin is a more potent inhibitor of the variant form of catechol-O-methyltransferase. Catechol-O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. It has been shown that COMT is genetically polymorphic with a wild-type and variant form where a valine has been substituted with a methionine. Several, but not all, epidemiological studies have shown that women, homozygous with the variant form, have an increased risk of developing breast cancer. Previously, we showed that 4-hydroxyequilenin (4-OHEN), a cytotoxic/genotoxic equine catechol estrogen metabolite, is both a substrate of COMT and an irreversible inhibitor of t...
Modification of standard freezing media to limit capacitation and maximise motility of frozen-thawed equine spermatozoa. To investigate cryopreservation-induced capacitation-like changes in equine spermatozoa frozen in three different media using chlortetracycline (CTC) fluorescence staining analysis. Methods: Semen collected from three stallions was diluted in one of three centrifugation media and, after centrifugation and removal of supernatant, extended in corresponding freezing media containing additional egg yolk, glycerol, lactose and Equex paste. The semen was frozen in 5 mL straws and the spermatozoa assessed for motility and membrane quality after thawing. Results: Following centrifugation, spermatozoa ...
In vitro effects of oxytetracycline on matrix metalloproteinase-1 mRNA expression and on collagen gel contraction by cultured myofibroblasts obtained from the accessory ligament of foals. To determine the effects of oxytetracycline on matrix metalloproteinase-1 (MMP-1) mRNA expression and collagen gel contraction by equine myofibroblasts in an effort to explain the mechanistic basis for the pharmacologic treatment of flexural deformities in foals. Methods: Cultured myofibroblasts from the accessory ligament (distal check ligament) of 6 foals. Methods: Collagen gel scaffolds seeded with equine myofibroblasts were cultured in individual culture dishes containing complete media (Dulbecco's modified Eagle medium with 10% fetal bovine serum) and oxytetracycline (0, 12.5, 25, or 75 m...
In vitro study of heat production during power reduction of equine mandibular teeth. To measure the amount of heat generated during 3 methods of equine dental reduction with power instruments. Methods: In vitro study. Methods: 30 premolar and molar teeth removed from mandibles of 8 equine heads collected at an abbatoir. Methods: 38-gauge copper-constantan thermocouples were inserted into the lingual side of each tooth 15 mm (proximal) and 25 mm (distal) from the occlusal surface, at a depth of 5 mm, which placed the tip close to the pulp chamber. Group-NC1 (n = 10) teeth were ground for 1 minute without coolant, group-NC2 (10) teeth were ground for 2 minutes without coolant, a...
Studies on motility and fertility of cooled stallion spermatozoa. This study on extended, cooled stallion spermatozoa aimed to compare the ability of three extenders to maintain sperm motility during 24 h of preservation, and to describe pregnancy and foaling rates after artificial insemination (AI) of stallion spermatozoa stored and transported in the extender chosen from the in vitro study. After 6 and 24 h of preservation, motility, both subjective and evaluated by the motility analyzer (total, progressive and rapid), was lower in non-fat, dried skim milk-glucose than in both other extenders: dried skim milk-glucose added to 2% centrifuged egg yolk, and u...
Cumulus-oocyte communications in the horse: role of the breeding season and of the maturation medium. Horse is a seasonal breeder and information on oocyte quality outside the breeding season is very limited. Ovaries obtained at the slaughterhouse are a convenient but often limited source of oocytes in this species. As the low quantity of ovaries leads to an intensive use of all available material, it would be useful to know whether ovaries collected during the non-breeding season are suitable for in vitro maturation (IVM). In an attempt to characterize the effect of season on oocyte quality, we investigated the permeability of the gap junctions (GJ) present between cumulus cells and oocytes b...
Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection. This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 degrees C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61-72 vs 23-2...
Plasma endotoxin concentration in horses: a methods study. Accurate determination of plasma endotoxin concentration is critical for ex vivo and in vitro cellular and molecular studies of endotoxemia in horses. However, reports are conflicting with respect to anticoagulant, handling, and sample preparation. Objective: The purpose of this study was to determine the effect of blood sample fraction and handling time on measurement of endotoxin concentration in horses. Methods: Whole blood, anticoagulated with 3.8% (0.12 M) sodium citrate (9:1), was collected from 5 healthy horses. Whole blood (WB), platelet-rich plasma (PRP), and platelet-poor plasma (PPP...
In vitro maturation and transfer of equine oocytes after transport of ovaries at 12 or 22 degrees C. Transportation of equine ovaries would allow shipment of oocytes for research purposes or transfer after the death of a valuable mare. The objective of this study was to compare two temperatures for maintaining ovaries during a transport interval of 18-24 h. The goal was to obtain pregnancies after transport of ovaries, maturation of oocytes in vitro, and transfer of oocytes. Each shipment was composed of ovaries four to seven mares collected from an abattoir. From each mare, one ovary was packaged at approximately 12 degrees C, and the other was packaged at approximately 22 degrees C. Upon ar...
Quantitative effect of tenorrhaphy on intrinsic vasculature of the equine superficial digital flexor tendon. To compare effects of the locking-loop suture pattern (LLP) and 3-loop pulley (3LP) suture pattern for tenorrhaphy on the intrinsic vasculature of the superficial digital flexor tendon (SDFT) of horses in vitro after surgery. Methods: 16 forelimbs obtained from 8 mature horses. Methods: Tenotomy and subsequent tenorrhaphy was performed in anesthetized horses. Following systemic administration of heparin, horses were euthanatized and the limbs were removed and placed under tension to load the flexor tendons. The intrinsic vasculature was then perfused with a mixture of barium sulfate and water....
A comparison of electroejaculation and epididymal sperm collection techniques in stallions. The purpose of this study was to evaluate 2 methods of semen collection that could be used as terminal procedures in stallions with irreparable conditions, such as fractures or colic. Electroejaculation was attempted under general anesthesia. Forty-eight hours later, the ponies were castrated and 2 different epididymal sperm collection techniques were attempted by using a flushing or floating method. Additionally, the effect of supplemental seminal plasma was evaluated. Experimentally, electroejaculation was found to be a safe but ineffective method of terminal semen collection. Viable sperm c...
Natural killer cell receptors in the horse: evidence for the existence of multiple transcribed LY49 genes. In rodents, the Ly49 family encodes natural killer (NK) receptors interacting with classical MHC class I molecules, whereas the corresponding receptors in primates are members of the killer cell immunoglobulin-like receptor (KIR) family. Recent evidence indicates that the cattle, domestic cat, dog, and pig have a single LY49 and multiple KIR genes, suggesting that predominant NK receptors in most non-rodent mammals might be KIR. Here, we show that the horse has at least six LY49 genes, five with an immunoreceptor tyrosine-based inhibition motif (ITIM) and one with arginine in the transmembrane...
Refined genome-wide comparative map of the domestic horse, donkey and human based on cross-species chromosome painting: insight into the occasional fertility of mules. We have made a complete set of painting probes for the domestic horse by degenerate oligonucleotide-primed PCR amplification of flow-sorted horse chromosomes. The horse probes, together with a full set of those available for human, were hybridized onto metaphase chromosomes of human, horse and mule. Based on the hybridization results, we have generated genome-wide comparative chromosome maps involving the domestic horse, donkey and human. These maps define the overall distribution and boundaries of evolutionarily conserved chromosomal segments in the three genomes. Our results shed further lig...
Current research and theories on the pathogenesis of acute laminitis in the horse. A large number of studies have been undertaken in recent years aimed at furthering our understanding of the complex mechanisms underlying the common and debilitating condition of acute laminitis in the horse. Many of these studies have either reinforced or cast doubt on previously held theories on the pathogenesis of this disease, while others have suggested new mechanisms which may play a key role in its development. This review seeks to put the current hypotheses into the context of this recent body of evidence. While a unifying theory may not yet seem to be achievable, this review demonstra...
Determination of passive mechanical properties of the superficial and deep digital flexor muscle-ligament-tendon complexes in the forelimbs of horses. To determine the relative contributions of the muscles, tendons, and accessory ligaments to the passive force-length properties of the superficial (SDF) and deep digital flexor (DDF) myotendinous complexes. Methods: 8 cadaveric forelimbs from 6 adult Thoroughbreds. Methods: In vitro, limb configurations during slack position and myotendinous lengths during subsequent axial loading of forelimbs were recorded before and after transection of accessory ligaments. Expressions were derived to describe the force-length behavior of each muscle, tendon, and accessory ligament-tendon unit; linear stiffn...
Molecular characterization and mutational screening of the PRKAG3 gene in the horse. The PRKAG3 gene encodes a muscle-specific isoform of the regulatory gamma subunit of AMP-activated protein kinase (AMPK). A major part of the coding PRKAG3 sequence was isolated from horse muscle cDNA using reverse-transcriptase (RT)-PCR analysis. Horse-specific primers were used to amplify genomic fragments containing 12 exons. Comparative sequence analysis of horse, pig, mouse, human, Fugu, and zebrafish was performed to establish the exon/intron organization of horse PRKAG3 and to study the homology among different isoforms of AMPK gamma genes in vertebrates. The results showed conclusively...
Japanese Society for Animal Reproduction: award for outstanding research 2002. Cryopreservation of follicular oocytes and preimplantation embryos in cattle and horses. Factors affecting sensitivity of preimplantation embryos and follicular oocytes to cryopreservation were analyzed in the equine and bovine species. (1) Survival of equine blastocysts after two-step freezing in the presence of glycerol as the cryoprotective agent (CPA) was influenced by development of the embryonic capsule. The use of ethylene glycol (EG) with sucrose as CPAs improved the post-thaw survival of blastocysts and made it possible to transfer the embryos into recipient mares without removing the CPAs. In addition, early blastocysts cryopreserved by vitrification could develop both i...
Influence of different collagen species on physico-chemical properties of crosslinked collagen matrices. Collagen-based scaffolds are appealing products for the repair of cartilage defects using tissue engineering strategies. The present study investigated the species-related differences of collagen scaffolds with and without 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS)-crosslinking. Resistance against collagenase digestion, swelling ratio, amino acid sequence, shrinkage temperature, ultrastructural matrix morphology, crosslinking density and stress-strain characteristics were determined to evaluate the physico-chemical properties of equine- and bovine-collagen...
Adhesion and proliferation of human dermal fibroblasts on collagen matrix. The purpose of this study was to evaluate adhesion and growth of human dermal fibroblasts on a 0.150 mm-thick matrix of reconstituted collagen isolated from horse tendon. Collagen was extracted and polymerized according to the standard procedures (Opocrin, Corlo, Modena, Italy). By light microscopy, the bottom surface of the matrix appeared linear and compact, whereas the superficial one was indented and less homogeneous. By scanning electron microscopy, the collagen fibrils had different diameters and the great majority of them was oriented parallel to the surface of the gel. By transmission ...
Peptide transport activity of the transporter associated with antigen processing (TAP) is inhibited by an early protein of equine herpesvirus-1. Equine herpesvirus-1 (EHV-1) downregulates surface expression of major histocompatibility complex (MHC) class I molecules on infected cells. The objective of this study was to investigate whether EHV-1 interferes with peptide translocation by the transporter associated with antigen processing (TAP) and to identify the proteins responsible. Using an in vitro transport assay, we showed that EHV-1 inhibited transport of peptides by TAP as early as 2 h post-infection (p.i). Complete shutdown of peptide transport was observed by 8 h p.i. Furthermore, pulse-chase experiments revealed that maturation...
Comparison of sensitivities of virus isolation, antigen detection, and nucleic acid amplification for detection of equine influenza virus. Four seronegative foals aged 6 to 7 months were exposed to an aerosol of influenza strain A/Equi/2/Kildare/89 at 10(6) 50% egg infective doses (EID(50))/ml. Nasopharyngeal swabs were collected for 10 consecutive days after challenge. Virus isolation was performed in embryonated eggs, and the EID(50) was determined for all positive samples. The 50% tissue culture infective dose was determined using Madin-Darby canine kidney (MDCK) cells. Samples were also tested by an in vitro enzyme immunoassay test, Directigen Flu A, and by reverse transcription-PCR (RT-PCR) using nested primers from the nucl...
Evaluation of viability and apoptosis in horse embryos stored under different conditions at 5 degrees C. The aim of this study was to evaluate the viability (percentage of dead cells) and the incidence of DNA fragmentation of horse embryos after storage in three different media at 5 degrees C for 6 and 24 h. Forty embryos were stored in Emcare Holding Solution for 6 and 24 h, in Hams'F10 or Vigro Holding Plus for 24 h at 5 degrees C (n = 9-10 per group) and 10 embryos were evaluated immediately after collection. First, embryos were stained, immediately after collection or following storage, to detect dead cells (DAPI) and, subsequently, DAPI-stained embryos were fixed and stained to detect DNA fr...
A dose titration of triamcinolone acetonide on insulin-like growth factor-1 and interleukin-1-conditioned equine cartilage explants. Previous in vitro pilot studies have defined a potentially beneficial effect of insulin-like growth factor-1 (IGF-1) and triamcinolone acetonide (TA) on interleukin-1 (IL-1)-conditioned equine cartilage. Furthermore, an optimal dose for IGF-1 treatment alone has been documented previously using the same test system as in the current project. Objective: To perform a dose titration of TA on IL-1-conditioned equine articular cartilage explants in the presence of an optimised IGF-1 dose, in order to optimise a triamcinolone concentration for use in combination with IGF-1 for future investigations....
Postnatal development of hepatic oxidative, hydrolytic and conjugative drug-metabolizing enzymes in female horses. Little is known about the effects of aging on the hepatic drug metabolizing capacity of horses despite the relatively long lifespan characterizing this species. A wide array of cytochrome P450 (CYP)-dependent monooxygenases, carboxylesterases and transferases were assayed in liver microsomes from 50 female horses in an age range between less than 1 year to over 12 years. Rather unexpectedly, both the CYP content and the activity of NADPH cytochrome c reductase rose as a function of age. Accordingly, a general increasing trend was recorded in the rate of the in vitro metabolism of the substrate...
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...
