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Topic:Biotechnology
Biotechnology in horses encompasses the application of biological techniques and tools to enhance equine health, performance, and reproduction. This field includes genetic engineering, cloning, and the development of vaccines and therapeutics tailored to equine physiology. Techniques such as gene editing and stem cell therapy are explored for their potential to address genetic disorders, improve tissue regeneration, and enhance disease resistance in horses. Additionally, advancements in reproductive biotechnology, such as artificial insemination and embryo transfer, contribute to genetic diversity and breeding efficiency. This page compiles peer-reviewed research studies and scholarly articles that investigate the applications, methodologies, and implications of biotechnology in equine science.
Altering equine corneal fibroblast differentiation through Smad gene transfer. To explore the impact of equine corneal fibroblast (ECF) to myofibroblast (ECM) differentiation by altering the expression of the Smad genes either individually or in combination. Specifically, we sought to examine the ECF differentiation after (a) silencing of Smad2, 3, and 4 profibrotic genes individually and (b) overexpression of antifibrotic Smad7 gene and in a combination with pro- and antifibrotic Smad genes. Methods: Equine corneal fibroblast primary cultures were generated as previously described. ECFs were transfected with individual plasmids which silenced gene expression of either S...
Identification of long non-coding RNA in the horse transcriptome. Efforts to resolve the transcribed sequences in the equine genome have focused on protein-coding RNA. The transcription of the intergenic regions, although detected via total RNA sequencing (RNA-seq), has yet to be characterized in the horse. The most recent equine transcriptome based on RNA-seq from several tissues was a prime opportunity to obtain a concurrent long non-coding RNA (lncRNA) database. This lncRNA database has a breadth of eight tissues and a depth of over 20 million reads for select tissues, providing the deepest and most expansive equine lncRNA database. Utilizing the intergen...
Evaluation of a telemetric gastrointestinal pill for continuous monitoring of gastrointestinal temperature in horses at rest and during exercise. OBJECTIVE To evaluate use of a telemetric gastrointestinal (GI) pill to continuously monitor GI temperature in horses at rest and during exercise and to compare time profiles of GI temperature and rectal temperature. ANIMALS 8 Standardbred horses. PROCEDURES Accuracy and precision of the GI pill and a rectal probe were determined in vitro by comparing temperature measurements with values obtained by a certified resistance temperature detector (RTD) in water baths at various temperatures (37°, 39°, and 41°C). Subsequently, both GI and rectal temperature were recorded in vivo in 8 horses over...
Expression of various sarcomeric tropomyosin isoforms in equine striated muscles. In order to better understand the training and athletic activity of horses, we must have complete understanding of the isoform diversity of various myofibrillar protein genes like tropomyosin. Tropomyosin (TPM), a coiled-coil dimeric protein, is a component of thin filament in striated muscles. In mammals, four TPM genes (TPM1, TPM2, TPM3, and TPM4) generate a multitude of TPM isoforms via alternate splicing and/or using different promoters. Unfortunately, our knowledge of TPM isoform diversity in the horse is very limited. Hence, we undertook a comprehensive exploratory study of various TPM i...
Comparison between chaotropic and detergent-based sample preparation workflow in tendon for mass spectrometry analysis. Exploring the tendon proteome is a challenging but important task for understanding the mechanisms of physiological/pathological processes during ageing and disease and for the development of new treatments. Several extraction methods have been utilised for tendon mass spectrometry, however different extraction methods have not been simultaneously compared. In the present study we compared protein extraction in tendon with two chaotropic agents, guanidine hydrochloride (GnHCl) and urea, a detergent, RapiGest™, and their combinations for shotgun mass spectrometry. An initial proteomic analysi...
Collection, processing and freezing of equine bone marrow cells. There is no consensus on aspects of equine bone marrow collection and processing. The study aimed to describe the collection of large volumes of bone marrow from horses of advanced age, with emphasis on bone marrow mononuclear cells (BMMCs) recovery and viability after cryopreservation. Fourteen horses, aged 3-24 years, were divided into three experiments. E1 studied the feasibility of collecting 200 mL from the sternums of horses of advanced age; E2 examined the number of cells obtained from the first and last syringe of each puncture; and E3 investigated the influence of heparin concentrati...
Efficacy of inactivation of viral contaminants in hyperimmune horse plasma against botulinum toxin by low pH alone and combined with pepsin digestion. Assuring viral safety of horse plasma-derived products is fundamental for ethical and regulatory reasons. We previously demonstrated the ability of pepsin digestion at low pH to inactivate West Nile and Sindbis viruses in horse plasma. The present study further examined the efficiency of pepsin digestion to inactivate four additional viruses: HSV-1 and BVDV (lipid-enveloped), BPV and Reo-3 (nonenveloped). These viruses were spiked into hyperimmunized horse plasma against botulinum toxin and subjected to low pH (3.2) alone or combined with pepsin digestion (1200 units/ml). Peptic digestion inac...
Species-specific identification of collagen components in Colla corii asini using a nano-liquid chromatography tandem mass spectrometry proteomics approach. Colla corii asini (CCA) is a protein-based traditional Chinese medicine made from donkey skins. Because it has the ability to nourish blood, its demand is increasing rapidly. The shortage of donkey skins increases the risk of the adulteration of CCA products with other animal skins. To ensure the drug efficacy and safety of CCA products, a proteomics technique was applied to reveal proteins in the skins of donkey, horse, cattle, and pig. Species-specific peptides for each animal species were predicted using bioinformatics, and their presence in the skins and gelatin samples was examined by nan...
The in vitro biocompatibility of d-(+) raffinose modified chitosan: Two-dimensional and three-dimensional systems for culturing of horse articular chondrocytes. The present study investigated the biocompatibility of chitosan films and scaffolds modified with d-(+)raffinose and their capability to support the growth and maintenance of the differentiation of articular chondrocytes in vitro. Primary equine articular chondrocytes were cultured on films and scaffolds of modified d-(+) raffinose chitosan. Their behavior was compared to that of chondrocytes grown in conventional bi- and three-dimensional culture systems, such as micromasses and alginate beads. Chitosan films maintained the phenotype of differentiated chondrocytes (typical round morphology) a...
Genotyping horse epithelial cells from fecal matter by isolation of polymerase chain reaction products. To show that application of the polymerase chain reaction (PCR) method modified for amplification of a low-copy number DNA samples, ie, the isolation of PCR products (IPCRp), would represent improvement in obtaining genotypes from a fecal DNA compared with previously used genotyping methods. Methods: The DNA from the horse fecal matter was extracted by modified Qiagen DNA Stool Mini Kit protocol. Following the extraction, the DNA genotypes from fecal samples were obtained by the most powerful PCR amplification method, the IPCRp. The IPCRp-based multiplex kit amplified biotin-labeled strands we...
RNA sample preparation applied to gene expression profiling for the horse biological passport. The improvement of doping control is an ongoing race. Techniques to fight doping are usually based on the direct detection of drugs or their metabolites by analytical methods such as chromatography hyphenated to mass spectrometry after ad hoc sample preparation. Nowadays, omic methods constitute an attractive development and advances have been achieved particularly by application of molecular biology tools for detection of anabolic androgenic steroids (AAS), erythropoiesis-stimulating agent (ESA), or to control human growth hormone misuses. These interesting results across different animal spe...
Defining the ABC of gene essentiality in streptococci. Utilising next generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality. Exposure of saturated mutant libraries to specific conditions and subsequent sequencing can be exploited to uncover gene essentiality relevant to the condition. Here we present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for Streptococcus equi subsp. equi, the causative agent of strangles in horses, for the first time. The gene essentiality data for this grou...
Equine performance genes and the future of doping in horseracing. A horse's success on the racetrack is determined by genetics, training and nutrition, and their translation into physical traits such as speed, endurance and muscle strength. Advances in genetic technologies are slowly explaining the roles of specific genes in equine performance, and offering new insights into the development of novel therapies for diseases and musculoskeletal injuries that cause early retirement of many racehorses. Gene therapy approaches may also soon provide new means to artificially enhance the physical performance of racehorses. Gene doping, the misuse of gene therapies f...
Development of a Sustained-Release Voriconazole-Containing Thermogel for Subconjunctival Injection in Horses. To determine in vitro release profiles, transcorneal permeation, and ocular injection characteristics of a voriconazole-containing thermogel suitable for injection into the subconjunctival space (SCS). In vitro release rate of voriconazole (0.3% and 1.5%) from poly (DL-lactide-co-glycolide-b-ethylene glycol-b-DL-lactide-co-glycolide) (PLGA-PEG-PLGA) thermogel was determined for 28 days. A Franz cell diffusion chamber was used to evaluate equine transcorneal and transscleral permeation of voriconazole (1.5% topical solution, 0.3% and 1.5% voriconazole-thermogel) for 24 hours. Antifungal activit...
A proteomic study of mesenchymal stem cells from equine umbilical cord. To the best of our knowledge, this is the first study describing the proteome of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs) in a global and functional manner. The aim of this work was to analyze the proteome of previously characterized UCIM-MSCs to determine protein abundance and classify the identified proteins according to Gene Ontology (GO) terms. Protein classification analysis according to biological process, molecular function and cellular component was performed using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification Syst...
Livestock metabolomics and the livestock metabolome: A systematic review. Metabolomics uses advanced analytical chemistry techniques to comprehensively measure large numbers of small molecule metabolites in cells, tissues and biofluids. The ability to rapidly detect and quantify hundreds or even thousands of metabolites within a single sample is helping scientists paint a far more complete picture of system-wide metabolism and biology. Metabolomics is also allowing researchers to focus on measuring the end-products of complex, hard-to-decipher genetic, epigenetic and environmental interactions. As a result, metabolomics has become an increasingly popular "omics" app...
Magnetic particle translation as a surrogate measure for synovial fluid mechanics. The mechanics of synovial fluid vary with disease progression, but are difficult to quantify quickly in a clinical setting due to small sample volumes. In this study, a novel technique to measure synovial fluid mechanics using magnetic nanoparticles is introduced. Briefly, microspheres embedded with superparamagnetic iron oxide nanoparticles, termed magnetic particles, are distributed through a 100μL synovial fluid sample. Then, a permanent magnet inside a protective sheath is inserted into the synovial fluid sample. Magnetic particles translate toward the permanent magnet and the percentage ...
Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes. The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previous...
An Ambulatory Electroencephalography System for Freely Moving Horses: An Innovating Approach. Electroencephalography (EEG) that has been extensively studied in humans presents also a large interest for studies on animal brain processes. However, since the quality of the recordings is altered by muscular activity, most EEG recordings on animals are obtained using invasive methods with deeply implanted electrodes. This requires anesthesia and can thus only be used in laboratory or clinical settings. As EEG is a very useful tool both for detecting brain alterations due to diseases or accidents and to evaluate the arousal and attentional state of the animal, it seemed crucial to develop a ...
Whole genome sequencing reveals a novel deletion variant in the KIT gene in horses with white spotted coat colour phenotypes. White spotting phenotypes in horses can range in severity from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for such phenotypes in horses. For the present study, we re-investigated a large horse family segregating a variable white spotting phenotype, for which conventional Sanger sequencing of the candidate genes' individual exons had failed to reveal the causative variant. We obtained whole genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This...
Platelet lysate obtained via plateletpheresis performed in standing and awake equine donors. Platelet preparations containing growth factors, attachment factors, and enzymes are appealing to enhance healing of injured tissues and as an alternative to xenogenic serum in cell culture media. Plateletpheresis is commonly used to collect platelets in human medicine but has not been validated in horses. Plateletpheresis to collect platelet concentrate was performed on six female, mixed breed, chemically restrained horses using commercially available apheresis equipment. Before and immediately after plateletpheresis, we performed physical examinations and collected blood for chemistry and co...
Isolation and characterization of equine native MSC populations. In contrast to humans in which mesenchymal stem/stromal cell (MSC) therapies are still largely in the clinical trial phase, MSCs have been used therapeutically in horses for over 15 years, thus constituting a valuable preclinical model for humans. In human tissues, MSCs have been shown to originate from perivascular cells, namely pericytes and adventitial cells, which are identified by the presence of the cell surface markers CD146 and CD34, respectively. In contrast, the origin of MSCs in equine tissues has not been established, preventing the isolation and culture of defined cell population...
Intelligence-based anti-doping from an equine biological passport. The move towards personalized medicine derived from individually focused clinical chemistry measurements has been translated by the human anti-doping movement over the past decade into developing the athlete biological passport. There is considerable potential for animal sports to adapt this model to facilitate an intelligence-based anti-doping system. Copyright © 2017 John Wiley & Sons, Ltd.
Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay. Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (IgA) in mucosal secretions of horses. Despite this, it is widely assumed that IgA is the predominant immunoglobulin on mucosal surfaces in the horse. Here, two new monoclonal antibodies (mAbs) against equine IgA, clones 84-1 and 161-1, were developed and characterized in detail. Both IgA mAbs specifically bound monomeric and dimeric equine IgA in different applications, such as Western blots and fluorescent bead-based assays. Cross-reactivity with other equine immunoglobulin isotypes was not observe...
Three-Dimensional Coculture of Meniscal Cells and Mesenchymal Stem Cells in Collagen Type I Hydrogel on a Small Intestinal Matrix-A Pilot Study Toward Equine Meniscus Tissue Engineering. Meniscal injuries are the most frequently encountered soft tissue injuries in the equine stifle joint. Due to the inherent limited repair potential of meniscal tissue, meniscal injuries do not only affect the meniscus itself but also lead to impaired joint homeostasis and secondary osteoarthritis. The presented study compares 3D coculture constructs of primary equine mesenchymal stem cells (MSC) and meniscus cells (MC) seeded on three different scaffolds-a cell-laden collagen type I hydrogel (Col I gel), a tissue-derived small intestinal matrix scaffold (SIS-muc) and a combination thereof-for ...
Effect of needle diameter on the viability of equine bone marrow derived mesenchymal stem cells. Mesenchymal stem cells (MSCs) are frequently delivered via needle injection for treatment of musculoskeletal injuries. The purpose of this study was to evaluate the effect of needle diameter on the viability of MSCs. Methods: Equine bone marrow-derived MSCs from 5 horses were suspended in PBS, and held at room temperature for 7 hours to mimic shipping conditions. Two replicate samples for each needle size (20, 22, 23, or 25-gauge [ga]) were aspirated into a 3 mL syringe and re-injected into the holding vial 3 times, to reproduce the resuspension of cells prior to injection in clinical cases. C...
Identification and Expression of Equine MER-Derived miRNAs. MicroRNAs (miRNAs) are single-stranded, small RNAs (21-23 nucleotides) that function in gene silencing and translational inhibition via the RNA interference mechanism. Most miRNAs originate from host genomic regions, such as intergenic regions, introns, exons, and transposable elements (TEs). Here, we focused on the palindromic structure of medium reiteration frequencies (MERs), which are similar to precursor miRNAs. Five MER consensus sequences (MER5A1, MER53, MER81, MER91C, and MER117) were matched with paralogous transcripts predicted to be precursor miRNAs in the horse genome (equCab2) and...
A foam model highlights the differences of the macro- and microrheology of respiratory horse mucus. Native horse mucus is characterized with micro- and macrorheology and compared to hydroxyethylcellulose (HEC) gel as a model. Both systems show comparable viscoelastic properties on the microscale and for the HEC the macrorheology is in good agreement with the microrheology. For the mucus, the viscoelastic moduli on the macroscale are several orders of magnitude larger than on the microscale. Large amplitude oscillatory shear experiments show that the mucus responds nonlinearly at much smaller deformations than HEC. This behavior fosters the assumption that the mucus has a foam like structure ...
Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during pronuclear development in equine zygotes produced by ICSI. Global epigenetic reprogramming is considered to be essential during embryo development to establish totipotency. In the classic model first described in the mouse, the genome-wide DNA demethylation is asymmetric between the paternal and the maternal genome. The paternal genome undergoes ten-eleven translocation (TET)-mediated active DNA demethylation, which is completed before the end of the first cell cycle. Since TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, the latter is postulated to be an intermediate stage toward DNA demethylation. The maternal genome, on the other ha...
Evaluation of equine oocyte developmental competence using polarized light microscopy. The purpose of this study was to observe -matured equine oocytes with an objective computerized technique that involves the use of a polarized light microscope (PLM) in addition to the subjective morphological evaluation obtained using a classic light microscope (LM). Equine cumulus-oocyte complexes (COCs, = 922) were subjected to different maturation times (24, 36 or 45 h), however, only 36-h matured oocytes were analyzed using CLM. The 36-h matured oocytes that reached maturity were parthenogenetically activated to evaluate the quality and meiotic competence. Average maturation perce...
