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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.
Effect of sperm extract injection volume, injection of PLCzeta cRNA, and tissue cell line on efficiency of equine nuclear transfer. We evaluated the effect of different activation methods on blastocyst development after equine nuclear transfer. All activation treatments were followed by incubation in 2 mM 6-dimethylaminopurine for 4 h. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 sec using a FemtoJet injection device, then treated with ionomycin. The blastocyst rate (9.8%) for 0.1-sec injection was significantly higher than that for 0.2 sec (0%) or 0.8 sec (1.4%). In Experiment 2, injection of murine PLCzeta cRNA before or after ionomycin treatment did not increase ...
Improved isolation protocol for equine cord blood-derived mesenchymal stromal cells. A robust methodology for the isolation of cord blood-derived multipotent mesenchymal stromal cells (CB-MSCs) from fresh umbilical cord blood has not been reported in any species. The objective of this study was to improve the isolation procedure for equine CB-MSCs. Methods: Pre-culture separation of red and white blood cells was done using either PrepaCyte?-EQ medium or Ficoll-Paque? PREMIUM density medium. Regular FBS and MSC-qualified FBS were compared for their ability to support the establishment of putative primary MSC colonies. Conclusions: Our results indicate that PrepaCyte-EQ medium i...
Detection of sex chromosome aneuploidy in equine spermatozoa using fluorescence in situ hybridization. The aim of our study was to diagnose aneuploidy in equine spermatozoa by multicolour fluorescence in situ hybridization (FISH) technique using specific molecular probes for equine sex chromosomes and autosome pair four (EGFR probe) labeled by different fluorochromes. These were applied on decondensed spermatozoa of four stallions. In total, more than 8800 sperm cells were examined. The total frequency of aberrant cells was 0.496%: aneuploidy of XX (0.135%), YY (0.023%), XY (0.102%), diploidy (0.057%), lack of sex chromosome (0.18%). In one stallion the ratio of normal X- and Y-bearing cells wa...
In vitro evaluation of differences in phase 1 metabolism of ketamine and other analgesics among humans, horses, and dogs. To investigate cytochrome P450 (CYP) enzymes involved in metabolism of racemic and S-ketamine in various species and to evaluate metabolic interactions of other analgesics with ketamine. Methods: Human, equine, and canine liver microsomes. Methods: An analgesic was concurrently incubated with luminogenic substrates specific for CYP 3A4 or CYP 2C9 and liver microsomes. The luminescence signal was detected and compared with the signal for negative control samples. Ketamine and norketamine enantiomers were determined by use of capillary electrophoresis. Results: A concentration-dependent decrease...
Comparison of equine tendon-, muscle-, and bone marrow-derived cells cultured on tendon matrix. To compare viability and biosynthetic capacities of cells isolated from equine tendon, muscle, and bone marrow grown on autogenous tendon matrix. Methods: Cells from 4 young adult horses. Methods: Cells were isolated, expanded, and cultured on autogenous cell-free tendon matrix for 7 days. Samples were analyzed for cell viability, proteoglycan synthesis, collagen synthesis, and mRNA expression of collagen type I, collagen type III, and cartilage oligomeric matrix protein (COMP). Results: Tendon- and muscle-derived cells required less time to reach confluence (approx 2 weeks) than did bone marr...
Risk of equine infectious anemia virus disease transmission through in vitro embryo production using somatic cell nuclear transfer. Prevention and regulation of equine infectious anemia virus (EIAV) disease transmission solely depend on identification, isolation, and elimination of infected animals because of lack of an effective vaccine. Embryo production via the somatic cell nuclear transfer (SCNT) technology uses oocytes collected mainly from untested animals, which creates a potential risk of EIAV transmission through infected embryos. The current review examines the risk of EIAV disease transmission through SCNT embryo production and transfer. Equine infectious anemia virus is a lentivirus from the family Retroviridae...
Mechanical recovery of inhibited cyathostomin larvae from equine intestinal tissue. The Stomacher is very widely used in food and medical research for extracting tissues. To determine whether nematode larvae were disrupted by the Stomacher, L3 larvae of Haemonchus contortus were homogenised for up to 40 min at full power but no larval disruption occurred. Therefore, tissue from the mucosa and submucosa of the caecum of horses collected from a licenced abattoir was treated to determine whether inhibited cyathostomin larvae could be extracted. The optimum time on full power for a 10-g sample was 20 min, and in three out of five caecal samples from different horses, significantl...
The effects of limb posture on relationships between in vitro radial hoof strain, load and joint angles. Radial strain in normal hooves has been found to vary with strain gauge location, limb posture and sample limb but reported magnitudes were considered to be low. More accurate measurement of radial strain may enhance the understanding of hoof function. Objective: To explore in vitro radial hoof strain in relation other kinetic and kinematic variables that may be related. Methods: Five normal forelimbs were removed at the proximal articular surface of the third metacarpal bone (McIII). The limbs were loaded using a modified Instron test machine. Six calibrated infrared cameras captured movement...
In vitro evaluation of metacarpophalangeal joint loading during simulated walk. Insight into the loading pattern of the articular cartilage surface during the complete stride is important as biomechanical factors play a pivotal role in the pathogenesis of joint trauma and osteoarthritis (OA). Objective: To determine the loading pattern in the equine MCP articulation in vitro during simulated walk. Methods: Eight cadaveric limbs from mature Dutch Warmblood horses were loaded in a pneumatic loading device in 6 different positions (A1-A6). The pressure distribution on the articular surface of the proximal phalanx (P1) was measured at 7 sites (S1-7) using intra-articularly pl...
Deformation of the equine pelvis in response to in vitro 3D sacroiliac joint loading. Sacroiliac joint injuries can cause poor performance; however, the interaction between pelvic mechanics and the sacroiliac joint is poorly understood. Objective: To measure pelvic displacement during 3D sacroiliac joint loading. Methods: Nine reflective triads were attached rigidly to bony prominences in sacropelvic specimens harvested from 14 horses for stereophotogrammetric analysis of triad displacements and joint kinematics. The sacrum was coupled to a load cell and mounted vertically within a material testing system (MTS). A pneumatic actuator was used to apply 90 Nm moments to the ischia...
Isolation, growth and differentiation of equine mesenchymal stem cells: effect of donor, source, amount of tissue and supplementation with basic fibroblast growth factor. Mesenchymal stem cells (MSC) are increasingly used as therapeutical aid for the orthopaedic injuries in the horse. MSC populate different tissues but the most commonly used for clinical purposes are isolated from bone marrow or adipose tissue. The first objective of this study was to investigate if the donor animal, the tissue of origin and the technique of isolation could influence the number of MSC available for transplantation after a short-term expansion. The second aim was to devise a culture system capable of increasing MSC lifespan and we tested the effect of basic fibroblast growth fac...
Equine versus bovine pericardium in transmeatal underlay myringoplasty. Many different grafting materials have been proposed in myringoplasty. The aim of this study was to evaluate the results obtained in transmeatal underlay myringoplasty using bovine and equine pericardium. The results were compared with those obtained by using autologous temporalis fascia. Methods: The study group consisted of 52 patients with tympanic perforation. Twenty-nine patients were randomly selected for treatment with bovine pericardium and 23 for equine pericardium. A group of 14 patients was treated with autologous temporalis fascia. Results: Closure of the perforation was achieved i...
Replication of equine herpesvirus type 1 in equine dermal cells transfected with Bam HI[G] restriction fragment of EHV-2 genome. In previous experiments, we have demonstrated that the presence of equine herpesvirus 2 (EHV-2) enhanced plaque formation in cell cultures infected with equine herpesvirus type 1. To determine whether a specific region of the EHV-2 genome is responsible for this effect, we have constructed a library of Bam HI fragments of the EHV-2 genome ligated into pcDNA plasmid. Equine dermal (ED) cell cultures were subsequently transfected with the constructs, passaged 5 times, tested for the presence of the plasmids and infected with EHV-1 at MOI = 0.01. Only in cultures transfected with the pcDNA/Bam HI...
Optimalization of fluorescence in situ hybridization conditions in mare oocytes and mouse embryos. The aim of the study was to optimize hybridization conditions of molecular probes specific for X sex chromosomes of the domestic horse in mare oocyte chromosomes. Mare oocytes, recovered from slaughterhouse ovaries by scraping the granulosa layer, were cultured in vitro. Metaphase II mature oocytes were treated with hypotonic solution and fixed, followed by hybridization of the molecular probe specific for the X chromosome ofthe domestic horse. Hybridization of probes specific for mouse heterosomes on mouse oocytes and early embryos was performed to verify the FISH technique. Of 438 oocytes an...
Synthesis and characterization of biologically active recombinant elk and horse FSH. The objective of this investigation was to clone and express the elk and horse common alpha-subunit and FSH beta-subunit cDNAs, and to produce recombinant FSH from both species in vitro. The RNAs extracted from elk and horse pituitary glands were reverse-transcribed and amplified by polymerase chain reaction. The cDNAs corresponding to both subunits of elk and horse were cloned into the expression vector pBudCE4.1 and transfected into CRL-9096 cells. Expression of both genes was determined in the transfected cells by Northern and Western blot analysis. Recombinant elk and horse FSH secreted in...
Stability and reproducibility of ADVIA 120-measured red blood cell and platelet parameters in dogs, cats, and horses, and the use of reticulocyte haemoglobin content (CH(R)) in the diagnosis of iron deficiency. Modern laser-based haematology analysers such as the ADVIA 120 have species-specific software and offer the possibility of assessing new haematological parameters. These parameters have yet to be evaluated, and as these analysers are often used in referral laboratories, it is important to know whether the values of haematological parameters change during sample transport. Therefore, samples of EDTA-anticoagulated blood from nine healthy dogs and EDTA- and citrate-anticoagulated blood from six healthy horses were collected and stored at room temperature for 72 and 48 hours, respectively. In can...
In vitro development of cyathostomin larvae from the third stage larvae to the fourth stage: morphologic characterization, effects of refrigeration, and species-specific patterns. A mixed population of equine cyathostomin (Nematoda, Strongyloidea) infective third stage larvae (L3) was cultured in vitro using a cell-free medium. Some L3 were cultured immediately after Baermann collection from fecal cultures, while others were kept in water at 4 degrees C for 7 days before initiating the in vitro cultures. Cultures were examined daily for viability. At days 2, 7, 14 and 21 larvae were collected for identification of developmental stage and morphological changes, using both light and scanning electron microscopy. Larvae were classified as early L3 (EL3), developing L3 (DL3...
In silico detection and characteristics of novel microRNA genes in the Equus caballus genome using an integrated ab initio and comparative genomic approach. The importance of microRNAs at the post-transcriptional regulation level has recently been recognized in both animals and plants. We used the simple but effective sequential method of first Blasting known animal miRNAs against the horse genome and then using the located candidates to search for novel miRNAs by RNA folding method in the vicinity (+ -500 bp) of the candidates. Here, a total of 407 novel horse miRNA genes including 354 mature miRNAs were identified, of these, 75 miRNAs were grouped into 32 families based on seed sequence identity. MiRNA genes tend to be present as clusters in som...
Differences in early osteogenesis and bone micro-architecture in anterior lumbar interbody fusion with rhBMP-2, equine bone protein extract, and autograft. To investigate the microstructural differences and responsible mechanisms in early bone formation in anterior lumbar interbody fusion (ALIF) in the spine using rhBMP-2 (INFUSE), equine bone protein extract (COLLOSS E) or autograft. Methods: Twelve Danish female landrace pigs underwent a 3-level ALIF procedure at L3-6. PEEK interbody cages packed with rhBMP-2, COLLOSS E, or autograft were inserted. The animals were divided into two groups of six, and observed for four and eight weeks postoperatively. MicroCT was performed for evaluation of microstructure of the bone within the cage. A mathemati...
Microsatellite loci in urine supernatant and stored samples from racehorses. To evaluate whether urine supernatant contains amplifiable DNA and to determine factors that influence genotyping of samples from racehorses after storage and transportation. Methods: 580 urine, 279 whole blood, and 40 plasma samples obtained from 261 Thoroughbreds and Standardbreds. Methods: Genomic DNA was isolated from stored blood and urine samples collected from racehorses after competition. Quantified DNA was evaluated to determine whether 5 equine microsatellite loci (VHL20, HTG4, AHT4, HMS6, and HMS7) could be amplified by use of PCR techniques. Fragment size of each amplified locus wa...
Viability and cell cycle analysis of equine fibroblasts cultured in vitro. This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and pro...
Equine thrombospondin II and secreted protein acidic and cysteine-rich in a model of normal and pathological wound repair. Wound healing in horses is complicated, particularly when wounds are on the limb. The objectives of this study were to clone equine thrombospondin II (THBS2) and secreted protein acidic and cysteine-rich (SPARC) cDNAs and to compare the spatiotemporal expression of mRNAs and proteins during repair of body and limb wounds. These molecules were targeted in view of their potential biological contribution to angiogenesis, which is exacerbated during the repair of limb wounds in horses. Cloning was achieved by screening size-selected cDNA libraries previously derived from 7-day-old wounds. Expressi...
Lectin-binding sites in isolated equine cumulus-oocyte complexes: differential expression of glycosidic residues in complexes recovered with compact or expanded cumulus. Equine cumulus-oocyte complexes (COCs) were analyzed by means of 13 lectins to evaluate their glycoconjugate patterns and to verify differences between COCs recovered with compact (Cp) and expanded (Exp) cumulus. Cumulus cells showed a similar staining pattern in both Cp and Exp COCs with all lectins used, except for a higher reactivity with SNA and GSA II in Cp COCs and SBA in Exp COCs. The zona pellucida (ZP) showed (1) uniform staining with MAL II, RCA(120), and SBA in both Cp and Exp COCs, (2) trilaminar binding pattern with WGA as well as higher Con A reactivity in the outer region of bot...
Horse bone marrow mesenchymal stem cells express embryo stem cell markers and show the ability for tenogenic differentiation by in vitro exposure to BMP-12. Mesenchymal stem cells (MSCs) have been recently investigated for their potential use in regenerative medicine. MSCs, in particular, have great potential, as in various reports they have shown pluripotency for differentiating into many different cell types. However, the ability of MSCs to differentiate into tendon cells in vitro has not been fully investigated. Results: In this study, we show that equine bone marrow mesenchymal stem cells (BM-MSCs), defined by their expression of markers such as Oct4, Sox-2 and Nanog, have the capability to differentiate in tenocytes. These differentiated cell...
Effect of dehydration prior to cryopreservation of large equine embryos. Cryopreservation of equine embryos>300microm in diameter results in low survival rates using protocols that work well for smaller equine embryos. These experiments tested the potential benefit of incorporating a dehydration step prior to standard cryopreservation procedures. Forty-six, day 7-8, grade 1, equine embryos 300-1350microm in diameter were subjected to one of the following treatments: (A) 2 min in 0.6M galactose, 10min in 1.5M glycerol, slow freeze (n=21); (B) 10min in 1.5M glycerol, slow freeze (n=15); (C) 2min in 0.6M galactose, 10min in 1.5M glycerol, followed by exposure to thaw ...
Association between myeloperoxidase concentration in equine frozen semen and post-thawing parameters. Despite improvement of techniques, semen of 20% of stallions remains unfreezable. Recent studies focused on the impact of reactive oxygen species and oxidant enzymes on semen characteristics. Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after cell lysis. It is responsible for the formation of hypochlorous acid, a strong oxidant agent, which could damage spermatozoa. The aim of this study was to determine the relation between MPO concentration and characteristics of frozen semen from stallions. Thirty-five straws from different s...
Expression and function of 5-HT7 receptors in smooth muscle preparations from equine duodenum, ileum, and pelvic flexure. In horses, gastrointestinal (GI) disorders occur frequently and cause a considerable demand for efficient medication. 5-Hydroxytryptamine receptors (5-HT) have been reported to be involved in GI tract motility and thus, are potential targets for treating functional bowel disorders. Our studies extend current knowledge on the 5-HT(7) receptor in equine duodenum, ileum and pelvic flexure by studying its expression throughout the intestine and its role in modulating contractility in vitro by immunofluorescence and organ bath experiments, respectively. 5-HT(7) immunoreactivity was demonstrated in ...
Prostaglandin H synthase Type 2 is differentially expressed in endometrium based on pregnancy status in pony mares and responds to oxytocin and conceptus secretions in explant culture. The equine embryo must signal its presence to the uterus for pregnancy to continue to term. Mobility of the conceptus throughout the uterus is crucial for its survival, and this action presumably permits the conceptus to transmit its antiluteolytic signal to the endometrium. Studies were completed to establish whether this unidentified antiluteolytic signal targets prostaglandin G/H synthase 2 (PGHS2), a rate limiting enzyme in converting arachidonic acid to prostaglandins (PGs). In the first study, quantitative RT-PCR was used to determine the relative abundance of PGHS2 mRNA in endometrium d...
Equine lumican (LUM) cDNA sequence and spatio-temporal expression in an experimental model of normal and pathological wound healing. The development of exuberant granulation tissue, a situation that in some ways resembles the human keloid, compromises both the aesthetic and functional outcomes of wound repair in horses. To help elucidate the underlying molecular mechanisms the spatio-temporal expression of lumican (LUM) mRNA and protein for their potential contributions to tissue remodelling of body and limb wounds, was examined in an established experimental model. Expression was studied in intact skin and in samples of 1-, 2-, 3-, 4- and 6-week-old wounds of the body and forelimb. Temporal gene expression was determined b...
Slow release antibiotics for treatment of septic arthritis in large animals. The search for an effective treatment for septic arthritis is ongoing. Current therapies are expensive since they require repeated joint lavage and long term antibiotic treatment. Local application of antimicrobial drugs is advantageous because high concentrations can be attained at the infection site, although repeated injections increase the risk of superinfection of the joint. Thus, slow release formulations, which have the advantage of local treatment yet single application of the drug, are appealing. Antibiotics used in slow release formulations are selected for tissue compatibility, an a...
