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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.
[The influence of centrifugation on quality and freezability of stallion semen]. The aim of the present study was to investigate the influence of various centrifugation methods on sperm loss and quality of frozen-thawed semen. From at a total of 8 Warmblood stallions of the National Stud Farm in Avenches, 3 ejaculates each were collected and seminal plasma was removed using 3 different centrifugation regimes. In method I (reference method) centrifugation occurred by a speed of 600 x g during 10 minutes. In method II 1000 x g was used during 2 minutes while in method III centrifugation was performed by 2000 x g during 2 minutes. After centrifugation 90%, of the supernatant ...
Quantification of Staphylococcus aureus adhesion to equine bone surfaces passivated with Plasmalyte and hyperimmune plasma. To quantify the adhesion of Staphylococcus aureus to 4 equine bone surfaces passivated in a balanced polyionic solution (Plasmalyte) or hyperimmune equine plasma (Polymune plasma). Methods: In vitro comparative study. Methods: Third metacarpal bone (MC3) surface explants from 9 equine cadavers. Methods: Approximately 1 cm(2) sections of periosteum were removed from MC3 and stapled to sterile stainless steel screens. Three bone surface explants were cut using a surgical saw to present 1 cm(2) surfaces of subperiosteal bone, cut cortical bone, or endosteum. Duplicate explants of each surface wer...
Effect of ovary holding temperature and time on equine granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology. The effects of ovary holding time and temperature on granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology were investigated through a series of experiments. Three experiments were performed to determine the effect of ovary holding time and temperature on granulosa cell apoptosis. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 2h, (2) at 30 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6 or 6-10h, and (3) granulosa cells were held for 0, 1, 2, 3, 5, 12 or 24h in M199 with Hank's salts at room temperature (suboptimal incubation). Granulosa cell DNA was analysed by ...
Expression of equine interleukin-18 by baculovirus expression system and its biologic activity. The equine interleukin-18 (IL-18) cDNA that contains the coding sequence was cloned and a recombinant baculovirus, named AcEIL-18, was constructed. The recombinant protein of the equine IL-18 was expressed by AcEIL-18 and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Insect cells infected with AcEIL-18 secreted a precursor IL-18 with 24 kilo dalton (kDa) into the culture supernatant. Western blot analysis showed that mature equine IL-18 about 18 kDa was also confirmed without co-expression of caspase-1. Culture supern...
Cumulus expansion, nuclear maturation and connexin 43, cyclooxygenase-2 and FSH receptor mRNA expression in equine cumulus-oocyte complexes cultured in vitro in the presence of FSH and precursors for hyaluronic acid synthesis. The aim of this study was to investigate cumulus expansion, nuclear maturation and expression of connexin 43, cyclooxygenase-2 and FSH receptor transcripts in equine cumuli oophori during in vivo and in vitro maturation in the presence of equine FSH (eFSH) and precursors for hyaluronic acid synthesis. Equine cumulus-oocyte complexes (COC) were cultured in a control defined medium supplemented with eFSH (0 to 5 micrograms/ml), Fetal Calf Serum (FCS), precursors for hyaluronic acid synthesis or glutamine according to the experiments. After in vitro maturation, the cumulus expansion rate was incr...
Identification of amino-acids in the alpha-subunit first and third loops that are crucial for the heterospecific follicle-stimulating hormone activity of equid luteinizing hormone/choriogonadotropin. To identify amino-acids in the alpha-subunit important for expression of heterospecific FSH activity of horse (e) LH/choriogonadotropin (CG) (eLH) and donkey (dk) LH/CG (dkLH) (FSH/LH ratio ten times higher for eLH than for dkLH); this FSH activity absolutely requires an equid (donkey or horse) alpha-subunit combined with an equid beta-LH subunit. Methods: Chimeric alpha-subunits possessing the first 63 amino-acids of the porcine (p) and the last 33 amino-acids of the donkey alpha-subunit (alphap-dk) and the inverse (alphadk-p) were constructed. Porcine-specific amino-acids were introduced by ...
Screening of the equine intestinal microflora for potential probiotic organisms. Probiotics have not been demonstrated to provide any beneficial health effects in horses, possibly because of improper selection of probiotic organisms. This study was designed to identify lactic acid bacteria of equine origin with predetermined beneficial properties which might make them useful as therapeutic probiotics. Objective: A small percentage of lactic acid bacteria that are native to the intestinal tract of horses possess properties that may be useful in the treatment and/or prevention of gastrointestinal disease in horses. Methods: Faecal samples were collected from healthy mature h...
In vitro and in vivo studies of homocysteine in equine tissues: implications for the pathophysiology of laminitis. Elevated plasma homocysteine (HCy) concentration is a risk factor for cardiovascular diseases associated with endothelial dysfunction, including the human digital ischaemic disease, Raynaud's phenomenon. Objective: HCy causes dysfunction of equine vascular endothelium and elevated plasma concentrations predispose to laminitis. Objective: To determine 1) the concentration of HCy in vitro, which inhibits equine vascular endothelial cell function and 2) any association between risk of laminitis and plasma HCy concentration. Methods: Endothelial function was studied by measuring endothelium-depend...
Equine laminitis: glucose deprivation and MMP activation induce dermo-epidermal separation in vitro. Acute laminitis is characterised by hoof lamellar dermal-epidermal separation at the basement membrane (BM) zone. Hoof lamellar explants cultured in vitro can also be made to separate at the basement membrane zone and investigating how this occurs may give insight into the poorly understood pathophysiology of laminitis. Objective: To investigate why glucose deprivation and metalloproteinase (MMP) activation in cultured lamellar explants leads to dermo-epidermal separation. Methods: Explants, cultured without glucose or with the MMP activator p-amino-phenol-mercuric acetate (APMA), were subject...
Evidence for vascular and enzymatic events in the pathophysiology of acute laminitis: which pathway is responsible for initiation of this process in horses? To date, there is a substantial amount of data to support the hypotheses that vascular and enzymatic changes are ongoing in experimental laminitis. Furthermore, there is substantial in vitro evidence that the enzymatic changes weaken the dermo-epidermal attachments leading to mechanical failure of the hoof-bone interface of the equine digit. However, investigators of both the vascular and enzymatic theories have, to date, been unable to substantiate the effects of these pathophysiological changes in vivo on laminar tissues of horses afflicted with experimentally induced or naturally acquired l...
Modulation of the cytokine responses in equine macrophages following TACE-inhibition. The detrimental effects of the pro-inflammatory cytokine TNF-alpha during equine acute abdominal disease are well known. Its pivotal role in many human diseases has led to various in-depth studies regarding its release mechanism, in particular by TNF-alpha converting enzyme (TACE). In this study we investigated the inhibitory effect of a TACE-inhibitor on cytokine release (TNF-alpha, IL-1alpha and IL-6) in three different cell models, including U937 cells, a recently established equine macrophage cell line, known as eCAS, and primary equine PBMC. The aim was to show the similarity of TNF-alpha...
Equine seminal plasma reduces sperm binding to polymorphonuclear neutrophils (PMNs) and improves the fertility of fresh semen inseminated into inflamed uteri. Seminal plasma (SP) is known to have immunosuppressive properties in several species. Equine SP has been reported to reduce or inhibit chemotaxis, phagocytosis and complement activity in vitro. The type and amount of the SP component that suppresses sperm-polymorphonuclear neutrophil (PMN) binding in vitro was determined, and the effect of such suppression on the fertility of mares inseminated in the presence of uterine inflammation, was analyzed. Sperm cells were suspended in either SP, semen extender or a mixture of both, and each was mixed with PMN-rich uterine secretions collected at 12 h ...
A simple fluorescence labeling method to visualize the three-dimensional arrangement of collagen fibers in the equine periodontal ligament. In order to display the collagen-fiber arrangement in the equine periodontal ligament an inexpensive and easy staining procedure with fluorescein was applied to paraffin sections. After fluorescein labeling a section was suitable for successful examination with three special microscopical systems: a) fluorescence microscopy b) phase contrast microscopy and c) polarized light microscopy. Collagen fibers were clearly displayed as compact structures in the fluorescence microscope. This distinct feature of the fluorescent image generated an almost three-dimensional impression of the fiber arrangem...
The effect of enrofloxacin on cell proliferation and proteoglycans in horse tendon cells. Fluoroquinolone antibiotics have been used widely in humans and domestic animals, including horses, because of their broad-spectrum bactericidal activity, and relative safety. The use of fluoroquinolones, however, is not without risk. Tendonitis and spontaneous tendon rupture have been reported in people during or following therapy with fluoroquinolones. We have studied the effects of enrofloxacin, a fluoroquinolone antibiotic used commonly in domestic animals, on tendon cell cultures established from equine superficial digital flexor tendons. Effects on cell proliferation and morphology were ...
Effect of growth medium on hydrophobicity of Staphylococcus epidermidis. The present study was undertaken to evaluate the effect of growth medium on expression of hydrophobicity of Staphylococcus epidermidis. A total of 24 hydrophobic isolates of S. epidermidis, determined by n-hexadecane adherence assay (HAA) earlier were included. Five different growth media: horse blood agar (HBA), brain heart infusion agar (BHIA), brain heart infusion broth (BHIB), tryptic soy broth (TSB) and proteose peptone broth (PPB) were used. All 24 isolates exhibited the reproducible hydrophobicity when grown on HBA; however, 20 (83.33%), 19 (79.16%), 15 (62.50%) and 13 (54.16%) isolates...
Cloning and sequencing of a cDNA expressing a ribosomal P0 peptide from Culicoides nubeculosus (Diptera). Insect bite dermal hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of Culicoides spp. and sometimes Simulium spp. The aim of the investigation presented here was to identify allergens causing IBH. A cDNA library expressing recombinant Culicoides nubeculosus proteins was screened using affinity-purified serum from an IBH-affected horse. Screening of the library resulted in identification of one immunoreactive clone. The sequence of the cDNA insert was determined and revealed a 600 bp insert with an open reading frame coding for a 78 amino acid long protein, called rCu...
Equine beta-defensin-1: full-length cDNA sequence and tissue expression. beta-Defensins are cysteine-rich endogenously produced antimicrobial peptides that play an important role in innate immune defense. Although, previous investigations have identified beta-defensins in several mammalian species, no reports have identified equine beta-defensins. Using a strategy of database searching for expressed sequence tags (EST) we identified putative expression of equine beta-defensins in hepatic tissue. Based on this information, sequence specific primers were designed for the equine gene enabling the identification of the full-length cDNA sequence of equine beta-defensin-...
Comparison of Ca2+ release and uptake characteristics of the sarcoplasmic reticulum in isolated horse and rabbit cardiomyocytes. Both the cardiac action potential duration (APD) (0.6-1 s) and resting heart rate (30-40 beats/min) in the horse are significantly different from humans and smaller mammals, including the rabbit. This would be anticipated to have consequences for excitation-contraction (EC) coupling and require adaptation of the individual processes involved. The sarcoplasmic reticulum (SR) is one of the main components involved in EC coupling. This study examines and compares the activity of this organelle in the horse with that of the rabbit. In particular, the study focuses on SR Ca2+ release via the Ca2+ r...
A biomechanical comparison of headless tapered variable pitch compression and ao cortical bone screws for fixation of a simulated midbody transverse fracture of the proximal sesamoid bone in horses. To compare mechanical properties and failure characteristics of 2 methods of fixation for repair of a transverse, midbody fracture of the proximal sesamoid bone (PSB): 4.5-mm AO cortical bone screw (AO) placed in lag fashion and 4/5-mm Acutrak (AT) self-compressing screw. Methods: An in vitro biomechanical evaluation of intact forelimb preparations and forelimb preparations with a simulated midbody PSB fracture stabilized by a bone screw. Methods: Sixteen paired and 8 unilateral cadaveric equine forelimbs. Methods: A midbody transverse osteotomy was created in the medial PSB of bilateral forel...
Tritrichomonas foetus: a scanning electron microscopy study of erythrocyte adhesion associated with hemolytic activity. The in vitro hemolytic activity of Tritrichomonas foetus was investigated. The parasite was tested against human erythrocytes of groups A, B, AB, and O, and against erythrocytes of nine adult animals of different species (the rabbit, rat, chicken, cat, dog, swine, horse, bovine, and sheep). The results showed that T. foetus strains (ATCC KV1, K, PAL, 5022, RJ, 90) did not present any hemolytic activity against any human erythrocyte group nor against rabbit, rat, chicken, cat, dog and swine erythrocytes. T. foetus strains, however, lysed horse, bovine, and sheep erythrocytes. No hemolysin relea...
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...
