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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.
Methods for equine preantral follicles isolation: quantitative aspects. The aim of this study was to test the use of mechanical and mechanical-enzymatic methods, saline solution (SS), and PBS solution for the manipulation and isolation of mare ovarian preantral follicles (PAFs). The ovaries were subjected to mechanical isolation (mixer) alone or in association with enzymatic digestion (collagenase). Incubation times of 10 and 20 min were employed. In the first group, 4.1 ± 4.9 PAFs were harvested with the mechanical-enzymatic method vs 71.1 ± 19.2 with the mechanical procedure, showing a significant difference between methods; using SS and PBS, these number...
Temporal trends in in vitro antimicrobial susceptibility patterns of bacteria isolated from foals with sepsis: 1979-2010. Monitoring the development of antimicrobial resistance is important for the rational selection of appropriate antimicrobial drugs to initiate treatment of foals with sepsis. Objective: To identify temporal trends in antimicrobial susceptibility patterns of bacteria isolated from foals with sepsis. Methods: Retrospective review of medical records. Methods: Foals aged <30 days with a diagnosis of sepsis, confirmed by culture of bacteria, were included. Susceptibility data, expressed as minimum inhibitory concentrations (MICs) (MIC50 , MIC90 , MIC range) and percent of isolates that were susce...
Indicators of replicative damage in equine tendon fibroblast monolayers. Superficial digital flexor tendon (SDFT) injuries of horses usually follow cumulative matrix microdamage; it is not known why the reparative abilities of tendon fibroblasts are overwhelmed or subverted. Relevant in vitro studies of this process require fibroblasts not already responding to stresses caused by the cell culture protocols. We investigated indicators of replicative damage in SDFT fibroblast monolayers, effects of this on their reparative ability, and measures that can be taken to reduce it. Results: We found significant evidence of replicative stress, initially observing consistent...
Frequency of resistance in obligate anaerobic bacteria isolated from dogs, cats, and horses to antimicrobial agents. Clinical specimens from dogs, cats, and horses were examined for the presence of obligate anaerobic bacteria. Of 4,018 specimens cultured, 368 yielded 606 isolates of obligate anaerobic bacteria (248 from dogs, 50 from cats, and 308 from horses). There were 100 specimens from 94 animals from which only anaerobes were isolated (25 dogs, 8 cats, and 61 horses). The most common sites tested were abdominal fluid (dogs and cats) and intestinal contents (horses). The most common microorganism isolated from dogs, cats, and horses was Clostridium perfringens (75, 13, and101 isolates, respectively). Th...
Biological application of laser induced breakdown spectroscopy technique for determination of trace elements in hair. Analysis of trace elements in mammalian hair has the potential to reveal retrospective information about an individual's nutritional status and exposure. As trace elements are incorporated into the hair during the growth process, longitudinal segments of the hair may reflect the body burden during growth. Using LIBS technique, Na, K, Ca, Mg, Si, Fe, Pb and Zn were detected in a single strand of horse hair. The results obtained through LIBS technique on hair samples were compared with the traditional technique (AAS) on digested acidified solution of the same samples. The effects of the experime...
Evaluation of the affinity of various species and strains of Staphylococcus to adhere to equine corneocytes. Meticillin-resistant Staphylococcus aureus (MRSA) strain USA 500 predominately colonizes horses and people working with them. Previous studies demonstrate that some Staphylococcus species exhibit higher affinity for corneocytes of specific mammalian species. Objective: The objective was to determine the relative affinities of various MRSA strains, meticillin-susceptible S. aureus (MSSA) strains and a meticillin-susceptible Staphylococcus pseudintermedius (MSSP) for equine corneocytes. We hypothesized that MRSA strain USA 500 would exhibit greater adhesion than other staphylococcal strains te...
[How aliphatic alcohols and ph affect reactional capability of the horse blood serum cholinesterase at its interaction with organophosphorus inhibitors]. There was studied action of aliphatic alcohols (ethanol, propanol, isopropanol, n-butanol, isobutanol, secbutanol, tretbetanol) and pH on various kinds of reactional capability the serum cholinesterase. At the alcohols-affected inhibition of the cholinesterase hydrolytic activity, the determining role was played not the total number carbon atoms in the alcohol molecule, but by the "effective length" of the carbohydrate chain. The fact that the presence of alcohols did not affect parameters of the reverse cholinesterase inhibition with onium ions tetramethylammonium and choline allows suggestin...
Ultrasound-guided injection of the maxillary nerve in the horse. Infiltration of the equine maxillary nerve with local anaesthetic can be useful for both diagnostic and surgical procedures. The deep location and proximity of the nerve to surrounding vascular and orbital structures make an accurate, complication-free injection a challenge using traditional techniques reliant upon surface anatomical landmarks. Objective: To develop an ultrasound-guided injection technique of the maxillary nerve in equine cadavers and to evaluate its efficacy and potential for complications in vivo. Methods: Descriptive cadaver anatomical and clinical study. Methods: The relev...
Establishment and characterization of a primary and a metastatic melanoma cell line from Grey horses. The Grey horse phenotype, caused by a 4.6 kb duplication in Syntaxin 17, is strongly associated with high incidence of melanoma. In contrast to most human melanomas with an early onset of metastasis, the Grey horse melanomas have an extended period of benign growth, after which 50% or more eventually undergo progression and may metastasize. In efforts to define changes occurring during Grey horse melanoma progression, we established an in vitro model comprised of two cell lines, HoMel-L1 and HoMel-A1, representing a primary and a metastatic stage of the melanoma, respectively. The cell lines ...
In vitro effects of lidocaine on contractility of circular and longitudinal equine intestinal smooth muscle. The purpose of the study was to compare the contractility-enhancing effects of lidocaine in equine jejunal circular (CSM) and longitudinal smooth muscle (LSM) in vitro. In previous studies, more pronounced effects of lidocaine were observed in ischaemia-reperfusion (IR) injured smooth muscle. Therefore in this study, effects were examined in both non-injured control tissues and tissues challenged by a defined, artificial IR injury. Isometric contractile performance of CSM and LSM, assessed by frequency (F), amplitude (A) and mean active force (MAF) of contractions, was defined as contractility...
The antimicrobial activity of honey against common equine wound bacterial isolates. Delayed healing associated with distal limb wounds is a particular problem in equine clinical practice. Recent studies in human beings and other species have demonstrated the beneficial wound healing properties of honey, and medical grade honey dressings are available commercially in equine practice. Equine clinicians are reported to source other non-medical grade honeys for the same purpose. This study aimed to assess the antimicrobial activity of a number of honey types against common equine wound bacterial pathogens. Twenty-nine honey products were sourced, including gamma-irradiated and no...
Role of tumor necrosis factor-α, interferon-γ and Fas-ligand on in vitro nitric oxide activity in the corpus luteum. Normal reproductive function involves the expression of inflammatory mediators. Regarding the corpus luteum (CL), cytokines promote the cross-talk between immune, vascular and steroidogenic cells, among others. Moreover, TNF, IFNG and FASL were shown to regulate equine CL establishment and regression. We hypothesized that cytokines action on equine CL may be mediated by nitric oxide (NO), through the regulation of endothelial NO synthase (eNOS) expression. TNF increased eNOS mRNA level and NO metabolite (nitrite) production during CL growth. Cytokines combined action (TNF+IFNG+FASL) promoted e...
Identification of a human monoclonal antibody to replace equine diphtheria antitoxin for treatment of diphtheria intoxication. Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (h...
Pharmacokinetics and thrombolytic effects of the recombinant tissue-type plasminogen activator in horses. To test the efficacy of the recombinant tissue-type plasminogen activator (rt-PA) alteplase in horses, the thrombolytic effect was tested in in vitro generated equine thrombi. The extent of lysis was determined by measuring the decrease in thrombi weight over a period of 4 hours. In vivo pharmacokinetics of alteplase were determined in 6 healthy horses. A single dose (1 mg/kg) was applied via intravenous infusion over a period of 30 minutes Coagulation-related variables, blood count and clinical parameters were taken before the treatment and until 48 h after treatment. In addition, plasma rt-P...
Evaluation of the conjunctival fungal flora and its susceptibility to antifungal agents in healthy horses in Switzerland. To characterize the conjunctival fungal flora and to determine the susceptibility of 2 isolated molds to antifungal drugs in samples of 64 healthy horses from The National Stud in Switzerland. Methods: Conjunctival cytobrush samples were collected from both eyes of 64 ophthalmologically normal horses in August 2012 and subsequently cultured on Sabouraud's agar medium. Growing fungi were identified and counted. Etests or broth microdilution tests for Aspergillus fumigatus and Eurotium amstelodami were carried out to determine antifungal drug sensitivity. These species had previously been detect...
Biochemical characterization of stallion prostasomes and comparison to their human counterparts. Release of nanometer-sized prostasomes into human and equine semen suggests essential functions in their relationships with sperm cells and the fertilization process. The two types of prostasomes displayed ultrastructural similarities, albeit the human prostasomes were somewhat larger than the stallion prostasomes. A high ratio of saturated fatty acids was characteristic for the two prostasome types. Electrophoretic separation systems revealed an equine prostasomal pattern different from that of human. The 21 distinctive low molecular weight protein spots in the 2D-gel (with no counterparts in...
Equine oviduct explant culture: a basic model to decipher embryo-maternal communication. Equine embryos remain for 6 days in the oviduct and thus there is a need for an in vitro model to study embryo-oviductal interactions in the horse, since this subtle way of communication is very difficult to analyse in vivo. Until now, no equine oviduct explant culture model has been characterised both morphologically and functionally. Therefore, we established a culture system for equine oviduct explants that maintained epithelial morphology during 6 days of culture, as revealed by light microscopy and transmission electron microscopy. We demonstrated the presence of highly differentiated, ta...
Induction of cytochrome P450 enzymes in primary equine hepatocyte culture. In this study, we established cell culture conditions for primary equine hepatocytes allowing cytochrome P450 enzyme (CYP) induction experiments. Hepatocytes were isolated after a modified method of Bakala et al. (2003) and cultivated on collagen I coated plates. Three different media were compared for their influence on morphology, viability and CYP activity of the hepatocytes. CYP activity was evaluated with the fluorescent substrate 7-benzyloxy-4-trifluoromethylcoumarin. Induction experiments were carried out with rifampicin, dexamethasone or phenobarbital. Concentration-response curves for...
In vitro mesenchymal trilineage differentiation and extracellular matrix production by adipose and bone marrow derived adult equine multipotent stromal cells on a collagen scaffold. Directed differentiation of adult multipotent stromal cells (MSC) is critical for effective treatment strategies. This study was designed to evaluate the capability of equine MSC from bone marrow (BMSC) and adipose tissue (ASC) on a type I collagen (COLI) scaffold to undergo chondrogenic, osteogenic and adipogenic differentiation and form extracellular matrix (ECM) in vitro. Following determination of surface antigen expression, MSC were loaded into scaffolds in a perfusion bioreactor and loading efficiency was quantified. Cell-scaffold constructs were assessed after loading and 7, 14 and 21 d...
Involvement of miRNAs in equine follicle development. Previous evidence from in vitro studies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to determine changes in the levels of these miRNAs in relation to follicle selection, maturation, and ovulation in the monovular equine ovary. In Experiment 1, follicular fluid was aspirated during ovulatory cycles from the dominant (DO) and largest subordinate (S) follicles of an ovulatory wave and the dominant (DA) follicle of a mid-cycle...
In vitro induction of functional allergen-specific CD4+ CD25high Treg cells in horses affected with insect bite hypersensitivity. Insect bite hypersensitivity (IBH) is a recurrent allergic dermatitis of horses with similarities to human atopic eczema, caused by bites of insects of the genus Culicoides. Previous studies suggested a dysregulated T cell tolerance to Culicoides allergen in IBH-affected horses. Objective: We have investigated whether the suppressive function of CD4(+) CD25(high) cells is impaired in IBH-affected horses and possible ways to restore it. Methods: CD4(+) CD25(-) cells sorted from peripheral blood mononuclear cells (PBMC) were stimulated with irradiated autologous PBMC pulsed with Culicoides or te...
Epidermal-like architecture obtained from equine keratinocytes in three-dimensional cultures. Despite the high prevalence of skin conditions in the horse, there is a dearth of literature on the culture and biology of equine skin cells, and this is partially attributable to the lack of suitable in vitro skin models. The objective of this study was to develop a three-dimensional (3D) culture system that would support the proliferation and differentiation of equine keratinocytes, similar to that observed in natural epidermis. Cell monolayers were obtained from explants of equine skin and serially passaged as highly pure keratinocyte populations (> 95% of cells), based on their express...
The involvement of growth hormone in equine oocyte maturation, receptor localization and steroid production by cumulus-oocyte complexes in vitro. The objectives of this study were to evaluate the effects of equine growth hormone (eGH) on nuclear and cytoplasmic maturation of equine oocytes in vitro, steroid production by cumulus cells, and expression and subcellular localization of eGH-receptors (eGH-R) on equine ovarian follicles. Cumulus-oocyte complexes (COCs) were recovered by aspirating follicles <30 mm in diameter from abattoir-derived ovaries. The COCs were morphologically evaluated and randomly allocated to be cultured in either a control maturation medium or supplemented with 400 ng/mL eGH, for 30 h at 38.5°C in air with 5%...
Effect of sex sorting on stallion spermatozoa: Heterologous oocyte binding, tyrosine phosphorylation and acrosome reaction assay. The interest on sex sorting by flow cytometry on the equine industry has been increasing over the years. In this work, three different tests were performed in order to evaluate the membrane status of sorted stallion spermatozoa: assessment of binding ability to porcine oocytes, evaluation of acrosome integrity after stimulation with A23187, and detection of tyrosine phosphorylation. These evaluations were made after incubation for 0h, 1.5h and 3h in a capacitating medium. Sorted stallion spermatozoa attached similarly to the porcine oocytes, when compared with control samples. Sorted spermatoz...
In vivo and in vitro evaluation of the effects of domperidone on the gastrointestinal tract of healthy horses. To determine the effects of domperidone on in vivo and in vitro measures of gastrointestinal tract motility and contractility in healthy horses. Methods: 18 adult horses and tissue samples from an additional 26 adult horses. Methods: Domperidone or placebo paste was administered to healthy horses in a 2-period crossover study. Gastric emptying was evaluated after oral administration of domperidone paste (1.1 or 5.0 mg/kg) or placebo paste by means of the acetaminophen absorption test in 12 horses. Frequency of defecation, weight of feces produced, fecal moisture, and stomach-to-anus transit ti...
Design of NK-2-derived peptides with improved activity against equine sarcoid cells. Equine sarcoid is a topically accessible model for the evaluation of anticancer peptides acting by physical membrane disruption avoiding the complexity of a systemic application. We aim at evaluating and improving natural peptides for host defence as lead structures, where we focus on the cationic and amphipathic peptide NK-2. Cytotoxicity tests, fluorescence microscopy and a chip-based biosensor, which enabled real-time monitoring of cell metabolism, were applied. Cancer cell killing was dynamic with an initial phase of increased cellular respiration, followed by membrane destruction. NK-2 wa...
Trypan blue/giemsa staining to assess sperm membrane integrity in salernitano stallions and its relationship to pregnancy rates. Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inse...
A comparison of three-dimensional culture systems to evaluate in vitro chondrogenesis of equine bone marrow-derived mesenchymal stem cells. To compare in vitro three-dimensional (3D) culture systems that model chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs). Methods: MSCs from five horses 2-3 years of age were consolidated in fibrin 0.3% alginate, 1.2% alginate, 2.5×10(5) cell pellets, 5×10(5) cell pellets, and 2% agarose, and maintained in chondrogenic medium with supplemental TGF-β1 for 4 weeks. Pellets and media were tested at days 1, 14, and 28 for gene expression of markers of chondrogenic maturation and hypertrophy (ACAN, COL2B, COL10, SOX9, 18S), and evaluated by histology (hematoxylin and eosin, Tolu...
Regulation of hypoxia-inducible factor-1α and related genes in equine digital lamellae and in cultured keratinocytes. Hypoxia-inducible factor-1α (HIF-1A) is an important protein in the regulation/induction of many genes in the cellular and tissue response to hypoxia and a central mediator in inflammatory signalling. As both hypoxia and inflammatory events are purported to occur in the lamellar epidermis in sepsis-related laminitis in the equid, HIF-1A may play a central role in this disease process. Objective: To assess the regulation of HIF-1A and HIF-1A-related genes in the equine keratinocyte in vitro and in the lamellar tissue of horses with sepsis-related laminitis. Methods: In vivo and in vitro experi...
In vitro evaluation of defined oligosaccharide fractions in an equine model of inflammation. Dietary supplementation with oligosaccharides has been proven to be beneficial for health in several mammalian species. Next to prebiotic effects resulting in a modulation of gut micro biota, immunomodulatory effects of oligosaccharides have been documented in vivo. Supplementation with defined oligosaccharide fractions has been shown to attenuate allergic responses and enhance defensive immune responses. Despite the accumulating evidence for immunomodulatory effects, very limited information is available regarding the direct mechanism of action of oligosaccharides. This study aims to elucidat...
