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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.
A defined medium supports changes consistent with capacitation in stallion sperm, as evidenced by increases in protein tyrosine phosphorylation and high rates of acrosomal exocytosis. Efficient in vitro capacitation of stallion sperm has not yet been achieved, as suggested by low sperm penetration rates reported in in vitro fertilization (IVF) studies. Our objectives were to evaluate defined incubation conditions that would support changes consistent with capacitation in stallion sperm. Protein tyrosine phosphorylation events and the ability of sperm to undergo acrosomal exocytosis under various incubation conditions were used as end points for capacitation. Sperm incubated 4-6h in modified Whitten's (MW) with the addition of 25 mM NaHCO3 and 7 mg/mL BSA (capacitating mediu...
Equine CTNNB1 and PECAM1 nucleotide structure and expression analyses in an experimental model of normal and pathological wound repair. Wound healing in horses is fraught with complications. Specifically, wounds on horse limbs often develop exuberant granulation tissue which behaves clinically like a benign tumor and resembles the human keloid in that the evolving scar is trapped in the proliferative phase of repair, leading to fibrosis. Clues gained from the study of over-scarring in horses should eventually lead to new insights into how to prevent unwanted scar formation in humans. cDNA fragments corresponding to CTNNB1 (coding for beta-catenin) and PECAM1, genes potentially contributing to the proliferative phase of repair,...
Expression and localization of the mu-opioid receptor (MOR) in the equine cumulus-oocyte complex and its involvement in the seasonal regulation of oocyte meiotic competence. The micro-opioid receptor (MOR) was identified in equine oocytes, cumulus and granulosa cells. By RT-PCR, a 441bp fragment was observed. By immunoblotting, a 65 kDa band was detected in samples of winter anestrous whereas in cells recovered in breeding season, two bands, 65 and 50 kDa, were found. The 65 kDa band was significantly more intense in winter anestrous specimens. In samples recovered in the breeding season, this band significantly decreased with the raise of follicle size and was heavier in compact oocytes and cumulus cells. The protein was localized on the oolemma and within the cy...
Endometrial explant culture for characterizing equine endometritis. Endometritis after insemination is ubiquitous in the horse and is associated with semen and/or bacteria in the uterus. In up to 40% of horses, inflammation persists causing infertility. An endometrial explant culture was developed to study uterine secretion of prostaglandin F(2alpha) (PGF(2alpha)) in response to physiological and pathological challenge. Methods: Uteri were collected from mares, the endometrium dissected and explants from the uterine body or horn cultured in William's or RPMI medium. The response of explants to oxytocin, semen or bacteria compared to untreated tissue was tested...
Evaluation of equine laminar vein function: harvesting, dissection and the use of functional methods to distinguish between veins and arteries. Pharmacological evaluation of the unique equine laminar microvasculature is crucial to understanding its role in health and in diseases such as laminitis. However, separating the distinctive characteristics of arterial versus venous components of this complex vascular network has previously proved to be extremely difficult. Encased in a hard hoof capsule, isolation of individual blood vessels presents a considerable challenge. Exacerbating this difficulty, the laminar venous network is adapted to sustain high intravascular pressures and consequently has thickened walls, making the normally str...
Comparison of computer assisted surgery with conventional technique for treatment of abaxial distal phalanx fractures in horses: an in vitro study. To (1) evaluate and compare computer-assisted surgery (CAS) with conventional screw insertion (conventional osteosynthesis [COS]) for treatment of equine abaxial distal phalanx fractures; (2) compare planned screw position with actual postoperative position; and (3) determine preferred screw insertion direction. Methods: Experimental study. Methods: Cadaveric equine limbs (n=32). Methods: In 8 specimens each, a 4.5 mm cortex bone screw was inserted in lag fashion in dorsopalmar (plantar) direction using CAS or COS. In 2 other groups of 8, the screws were inserted in opposite direction. Precisi...
Establishment and characterization of equine fibroblast cell lines transformed in vivo and in vitro by BPV-1: model systems for equine sarcoids. It is now widely recognized that BPV-1 and less commonly BPV-2 are the causative agents of equine sarcoids. Here we present the generation of equine cell lines harboring BPV-1 genomes and expressing viral genes. These lines have been either explanted from sarcoid biopsies or generated in vitro by transfection of primary fibroblasts with BPV-1 DNA. Previously detected BPV-1 genome variations in equine sarcoids are also found in sarcoid cell lines, and only variant BPV-1 genomes can transform equine cells. These equine cell lines are morphologically transformed, proliferate faster than parental ...
Role of intracellular kinases in the regulation of equine eosinophil migration and actin polymerization. Inappropriately activated eosinophils can contribute to disease pathogenesis and intracellular signalling pathways that regulate functional responses may represent a therapeutic target. Little is known about intracellular signalling in equine eosinophils and this study examined the role of phospholipase C (PLC) and a range of protein kinases on responses to histamine and CCL11. Histamine (10(-4) M) or CCL11 (5.6 x 10(-9) M)-induced actin polymerization, migration and superoxide production by eosinophils from healthy horses were compared in the presence and absence of selective kinase inhibitor...
Characterization of agonist-induced endothelium-dependent vasodilatory responses in the vascular bed of the equine digit. The role of endothelium-derived relaxing factors was studied in the regulation of vascular responses in the Krebs perfused equine isolated digit. Perfusion pressure was recorded in response to bolus doses of 5-hydroxytryptamine (6 nmol) alone or co-administered with carbachol (CCh; 0.2 micromol), bradykinin (BK; 0.2 nmol), substance P (SP; 0.2 nmol) or sodium nitroprusside (SNP; 0.2 micromol). N(omega)-Nitro-L-Arginine methyl ester hydrochloride (L-NAME; 300 microm) caused partial but significant inhibition of CCh-induced vasodilatory response, whereas BK and SP-induced responses were resistan...
In vitro efficacy of an ophthalmic drug combination against corneal pathogens of horses. To evaluate the in vitro efficacy of an ophthalmic drug combination against common corneal pathogens of horses. Methods: Representative isolates of 3 bacterial and 2 fungal corneal pathogens of horses. Methods: Pathogens were subjected to minimum inhibitory concentration (MIC) testing of a drug combination that consisted of equal volumes of natamycin 3.33%, tobramycin 0.3%, cefazolin 5.5%, and equine serum. Proteinase inhibitory activity of the drug combination was assessed by use of a fluorescence microplate assay with gelatin and collagen I as substrates. The MICs of the drug combination wer...
In vitro cell compatibility and antibacterial activity of microencapsulated doxycycline designed for improved localized therapy of septic arthritis. For the treatment of septic arthritis in large animals, the local application of antibiotics as a slow release system may be an appropriate means to reach high local bioactivity and low systemic side effects and drug residues. In this study, doxycycline microspheres were developed and tested in vitro for their drug-release properties, suitability for intra-articular application and antimicrobial activity. Methods: The development of a slow release system was achieved by microencapsulation of the drug into poly(lactide-co-glycolide) microspheres by a novel ultrasonic atomization method. Drug el...
Ultrasound-guided periarticular injections of the sacroiliac region in horses: a cadaveric study. The traditional techniques for injection of the sacroiliac (SI) region are based on external landmarks. Because of the depth of the SI joint and pathological modifications, SI injections are sometimes challenging in horses. Objective: An ultrasound-guided techniques would allow placement of the needle without depending on external landmarks. Methods: Fourteen pelvic specimens were isolated from mature horses. A 20 cm bent spinal needle was positioned with ultrasonographic guidance under both iliac wings aiming for SI joints using 5 approaches: cranial, craniomedial, medial and 2 caudal approac...
Hypertrophy and physiological death of equine chondrocytes in vitro. Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes. Objective: To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo. Methods: Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). Th...
In vitro evaluation of a single injection technique for diagnostic analgesia of the proximal suspensory ligament of the equine pelvic limb. To examine the anatomic relationship of the deep branch of the lateral plantar nerve (DBLPN) with structures of the proximal metatarsal region, and to define the anatomic location of a solution injected using a single injection technique for diagnostic analgesia of the proximal suspensory ligament (PSL). Methods: Descriptive study. Methods: Cadaveric equine pelvic limbs (n=29). Methods: The proximal metatarsal region of both pelvic limbs (n=5 horses, 10 limbs) was dissected and measurements were made of anatomic structures relative to the head of the 4th metatarsal bone (HMT4). A single inject...
Platelet activation in ponies with airway inflammation. Platelet activation occurs in human obstructive airway diseases and in laboratory animal models. However, there is limited evidence that platelets may be involved in equine recurrent airway obstruction (RAO) and other inflammatory diseases. This study investigated whether platelet activation also occurred in RAO. Objective: Platelet function is altered in ponies with active RAO. This alteration can be detected ex vivo by measuring platelet adhesion. Methods: An in vitro platelet adhesion assay measuring acid phosphatase (AcP) activity colorimetrically was adapted for use with equine platelets ...
In vitro biomechanical comparison of equine proximal interphalangeal joint arthrodesis techniques: prototype equine spoon plate versus axially positioned dynamic compression plate and two abaxial transarticular cortical screws inserted in lag fashion. To compare in vitro monotonic biomechanical properties of an equine spoon plate (ESP) with an axial 3-hole, 4.5 mm narrow dynamic compression plate (DCP) using 5.5 mm cortical screws in conjunction with 2 abaxial transarticular 5.5 mm cortical screws (DCP-TLS) inserted in lag fashion for equine proximal interphalangeal (PIP) joint arthrodesis. Methods: Paired in vitro biomechanical testing of 2 methods of stabilizing cadaveric adult equine forelimb PIP joints. Methods: Cadaveric adult equine forelimbs (n=18 pairs). Methods: For each forelimb pair, 1 PIP joint was stabilized with an ESP (8 hole...
Phosphodiesterase isoenzymes in equine platelets and their influence on platelet adhesion. To determine the phosphodiesterase (PDE) isoenzymes in equine platelets and evaluate their influence on platelet adhesion. Methods: Platelets obtained from healthy New Forest Pony geldings that ranged from 12 to 20 years of age (mean +/- SEM, 17.3 +/- 1.1 years). Methods: PDE isoenzyme activity in equine platelets was determined by use of a 2-step radioactive assay. Functional importance of PDE isoenzymes was established by use of selective inhibitors in a colorimetric adhesion assay. Results: PDE1, PDE2, PDE3, and PDE5 and small amounts of PDE4 were found in equine platelets. Inhibition of PD...
Loss of expression of protein kinase a regulatory subunit 1alpha in pigmented epithelioid melanocytoma but not in melanoma or other melanocytic lesions. Pigmented epithelioid melanocytoma (PEM) is a recently described entity comprising most cases previously described as "animal-type melanoma" and epithelioid blue nevus (EBN) occurring in patients with the multiple neoplasia syndrome Carney complex (CNC). Mutations of the protein kinase A regulatory subunit type 1alpha (R1alpha) (coded by the PRKAR1A gene) are found in more than half of CNC patients. In this study, we investigated whether PEM and EBN are related at the molecular level, and whether changes in the PRKAR1A gene status and the expression of the R1alpha protein may be involved in th...
Effects of in vitro exposure to hay dust on expression of interleukin-17, -23, -8, and -1beta and chemokine (C-X-C motif) ligand 2 by pulmonary mononuclear cells isolated from horses chronically affected with recurrent airway disease. To examine effects of in vitro exposure to solutions of hay dust, lipopolysaccharide (LPS), or beta-glucan on cytokine expression in pulmonary mononuclear cells isolated from healthy horses and horses with recurrent airway obstruction (RAO). Methods: 8 RAO-affected and 7 control horses (experiment 1) and 6 of the RAO-affected and 5 of the control horses (experiment 2). Methods: Bronchoalveolar lavage cells were isolated from horses that had been stabled and fed dusty hay for 14 days. Pulmonary mononuclear cells were incubated for 24 (experiment 1) or 6 (experiment 2) hours with PBS solution or...
The horse genome project–sequence based insights into male reproductive mechanisms. The growing knowledge on physiology, cell biology and biochemistry of the reproductive organs has provided many insights into molecular mechanisms that are required for successful reproduction. Research directed at the investigation of reproduction physiology in domestic animals was hampered in the past by a lack of species-specific genomic information. The genome sequences of dog, cattle and horse have become publicly available in 2005, 2006 and 2007 respectively. Although the gene content of mammalian genomes is generally very similar, genes involved in reproduction tend to be less conserved...
Equine coronavirus induces apoptosis in cultured cells. Equine coronavirus (ECoV) was first isolated from a diarrheic foal and was found genetically similar to group II coronaviruses. However, its pathological characteristics were not adequately investigated. In our preliminary in vitro investigation, ECoV-induced cell death was observed in bovine kidney-derived MDBK cells. Based on this finding, we investigated whether the ECoV-induced CPE was apoptosis. Following ECoV infection, MDBK cells showed morphological changes such as cell rounding and detachment from the culture surface. Moreover, syncytium formation was observed as the other type of cyt...
Equine infectious anemia virus entry occurs through clathrin-mediated endocytosis. Entry of wild-type lentivirus equine infectious anemia virus (EIAV) into cells requires a low-pH step. This low-pH constraint implicates endocytosis in EIAV entry. To identify the endocytic pathway involved in EIAV entry, we examined the entry requirements for EIAV into two different cells: equine dermal (ED) cells and primary equine endothelial cells. We investigated the entry mechanism of several strains of EIAV and found that both macrophage-tropic and tissue culture-adapted strains utilize clathrin-coated pits for entry. In contrast, a superinfecting strain of EIAV, EIAV(vMA-1c), utilizes ...
Modeling trophoblast differentiation using equine chorionic girdle vesicles. The chorionic girdle of the equine conceptus is comprised of specialized trophoblast cells which, at day 36-38 of equine pregnancy, gain an invasive phenotype and invade the endometrium to form endometrial cups. Studies of equine endometrial cups remain difficult to perform because of the invasive techniques required to obtain cup tissue and because sampling requires termination of the pregnancy. In this study we developed a system to model trophoblast differentiation and trophoblast-immune interactions in vitro and in vivo. We utilized a method of culturing chorionic girdle pieces in serum-fr...
Lactobacillus hayakitensis sp. nov., isolated from intestines of healthy thoroughbreds. Two strains, KBL13(T) and GBL13, were isolated as one of intestinal lactobacilli from the faecal specimens from different thoroughbreds of the same farm where they were born in Hokkaido, Japan. They were Gram-positive, facultatively anaerobic, catalase-negative, non-spore-forming and non-motile rods. KBL13(T) and GBL13 homofermentatively metabolize glucose, and produce lactate as the sole final product from glucose. The 16S rRNA gene sequence, DNA-DNA hybridization, DNA G+C content and biochemical characterization indicated that these two strains, KBL13(T) and GBL13, belong to the same species...
Analysis of factor VIII mediated suppression of lentiviral vector titres. Effective gene therapy for haemophilia A necessitates a vector system that is not subject to a pre-existing immune response, has adequate coding capacity, gives long-term expression and preferably can target non-dividing cells. Vector systems based on lentiviruses such as equine infectious anaemia virus (EIAV) fulfil these criteria for the delivery of factor VIII (FVIII). We have found that B domain-deleted (BDD) FVIII protein inhibits functional viral particle production when co-expressed with the EIAV vector system. Although particle numbers (as measured by reverse transcriptase activity) ar...
A soluble secreted glycoprotein (eCLCA1) is overexpressed due to goblet cell hyperplasia and metaplasia in horses with recurrent airway obstruction. The equine putative chloride channel protein eCLCA1 is thought to be critically involved in the pathogenesis of recurrent airway obstruction (RAO) via modulation of the hydration of airway mucins. A recent study revealed a strong increase of eCLCA1 messenger ribonucleic acid (mRNA) in the lungs of horses with RAO. In this study, eCLCA1 protein and mRNA expression were quantified in airway goblet cells of 9 horses affected with RAO and 9 control horses by using immunohistochemistry and laser microdissection followed by real-time quantitative reverse transcription polymerase chain reaction, resp...
Effects of oxygen exposure and gentamicin on stallion semen stored at 5 and 15 degrees C. This study was undertaken to investigate the effects of storage of stallion semen in a defined milk protein extender at 5 and 15 degrees C under either anaerobic or aerobic conditions, with or without addition of the antibiotic gentamicin. Semen samples were collected from eight fertile stallions and stored for 96 h (day 0-4) and assessed daily for motility, velocity and membrane integrity (viability) using a CASA system. Samples for bacteriology assessment were taken on day 2 of storage. No significant (p > 0.05) differences in motility, velocity or viability were observed between treatmen...
Tissue engineering: chondrocyte culture on type 1 collagen support. Cytohistological and immunohistochemical study. The scope of our study is to evaluate the possibility of cultivating and expanding human chondrocytes and seeding them on pure equine type I collagen support. Our results show that human articular cartilaginous cells can multiply and grow on type I collagen substrate with production of extracellular matrix. This type of chondrocyte culture on a support can be used for repairing cartilaginous lesions since they show a correct morphology (evaluated by cytological and histological methods) and a suitable differentiation and phenotype as shown by Alcian PAS staining to indicate the presence of muc...
Development of an in vitro three dimensional loading-measurement system for long bone fixation under multiple loading conditions: a technical description. The purpose of this investigation was to design and verify the capabilities of an in vitro loading-measurement system that mimics in vivo unconstrained three dimensional (3D) relative motion between long bone ends, applies uniform load components over the entire length of a test specimen, and measures 3D relative motion between test segment ends to directly determine test segment construct stiffness free of errors due to potting-fixture-test machine finite stiffness.Intact equine cadaveric radius bones, which were subsequently osteotomized/ostectomized and instrumented with bone plates were su...
Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test. The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia col...
