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.
Construction and Application of an In-House Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Database Specific to Bacteria From Horses. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is used for identification of bacterial species isolated from horses. However, because of insufficiencies in the reference database, some bacterial species isolated from horses are difficult to identify with MALDI-TOF MS, and enriching the databases is expected to enhance the accuracy of MALDI-TOF MS identification. Here we created an in-house database including 271 bacterial isolates from horses. Furthermore, we used an enhanced database (our in-house database plus a commercially provided database) to ...
Development of a suitable manufacturing process for production of a bioactive recombinant equine chorionic gonadotropin (reCG) in CHO-K1 cells. Equine chorionic gonadotropin (eCG) is a heterodimeric glycoprotein hormone produced by pregnant mares that has been used to improve reproductive performance in different domestic species. Several strategies to produce the hormone in a recombinant way have been reported; nevertheless, no approach has been able to produce a recombinant eCG (reCG) with significant in vivo bioactivity or in sufficient quantities for commercial purposes. For this reason, the only current product available on the market consists of partially purified preparations from serum of pregnant mares (PMSG). Herein, we des...
Recommendations for Ensuring Good Welfare of Horses Used for Industrial Blood, Serum, or Urine Production. Various pharmaceutical products have been derived from horse blood and urine for over a century. Production of biologics and therapeutics from these samples is a niche industry and often occurs in regions with little regulation or veterinary oversight. To ensure good welfare of horses maintained for these purposes, guidance has been developed to support the industry.
Development of a constant pressure perfused ex vivo model of the equine larynx. Distal axonopathy is seen in a broad range of species including equine patients. In horses, this degenerative disorder of the recurrent laryngeal nerve is described as recurrent laryngeal neuropathy (RLN). The dysfunctional innervation of the cricoarytenoideus dorsalis muscle (CAD) leads to a loss of performance in affected horses. In general, ex vivo models of the larynx are rare and for equine patients, just one short report is available. To allow for testing new therapy approaches in an isolated organ model, we examined equine larynges in a constant pressure perfused setup. In order to chec...
Screening and detection of chromosomal copy number alterations in the domestic horse using SNP-array genotyping data. Chromosomal abnormalities are a common cause of infertility in horses. However, they are difficult to detect using automated methods. Here, we propose a simple methodology based on single nucleotide polymorphism (SNP)-array data that allows us to detect the main chromosomal abnormalities in horses in a single procedure. As proof of concept, we were able to detect chromosomal abnormalities in 33 out of 268 individuals, including monosomies, chimerisms, and male and female sex-reversions, by analyzing the raw signal intensity produced by an SNP array-based genotyping platform. We also demonstrat...
ANCHOR-tagged equine herpesvirus 1: A new tool for monitoring viral infection and discovering new antiviral compounds. Equine herpesvirus 1 (EHV-1) is a causative agent of respiratory disorders, abortion and myeloencephalopathy in horses and has an important impact on equine health and economy. Several bacterial artificial chromosomes have already been developed and enabled identification and functional characterization of EHV-1 genes. Unfortunately, little is known about its replication. Here, the ANCHOR system was inserted by targeted homologous recombination into the equine herpesvirus genome. This insertion led to the conversion of EHV-1 DNA to auto-fluorescent spots easily detectable by fluorescence micro...
The use of electrical impedance tomography (EIT) to evaluate pulse rate in anaesthetised horses. Electrical impedance tomography (EIT) provides clinically useful lung images; however, it would be an advantage to extract additional cardiovascular information from the data. The aim of this study was to evaluate if cardiac-related changes measured by EIT can be used to measure pulse rate (PR) under physiological as well as high and low blood pressure states in anaesthetised horses. Electrical impedance tomography data and PR from seven horses anaesthetised in dorsal recumbency were recorded over 1 min during mechanical ventilation and 1 min of apnoea. Data were collected at four measurement ...
Adeno-Associated Virus-Mediated Overexpression of Interleukin-10 Affects the Immunomodulatory Properties of Equine Bone Marrow-Derived Mesenchymal Stem Cells. Joint injury can cause posttraumatic inflammation, which if severe enough can lead to posttraumatic osteoarthritis (PTOA), a progressive and debilitating condition. Posttraumatic inflammation is characterized by an influx of T lymphocytes and upregulation of inflammatory cytokines and degradative enzymes by activated chondrocytes and synoviocytes. Intra-articular bone marrow-derived mesenchymal stem cell (BM-MSC) injection for the treatment of osteoarthritis (OA) has been of interest due to the immunomodulatory properties of these cells. Interleukin (IL)-10, a potent immunomodulatory cytokine,...
Development and performance of an automated fecal egg count system for small ruminant strongylids. An automated equine fecal egg count test, known as the Parasight System, was modified for use with small ruminants. Modifications included the introduction of a short centrifugation step in a floatation medium, an adjustment in pre-test sample filtering, and training of an image analysis-based egg counting algorithm to recognize and enumerate trichostrongylid eggs. In preliminary assessments, the modified method produced trichostrongylid egg counts comparable to manual McMaster analyses of the same samples from both ovine and caprine sources. The coefficient of determination (R) for the linear...
Preparation and evaluation of virus-like particle vaccine against H3N8 subtype equine influenza. To prevent and control H3N8 subtype equine influenza, we prepared virus-like particles (VLPs) comprising the HA, NA and M1 proteins of H3N8 equine influenza virus (EIV) through the insect cell-baculovirus expression system. The results of Western blot and hemagglutination analyses demonstrated that the constructed VLPs comprising HA, NA and M1 proteins have good hemagglutination activity. Immunoelectron microscope revealed that the VLPs share similar morphology and structure with natural virus particles. The hyperimmune serum from horses immunized with the VLPs were injected into mice by means...
Development and characterization of two equine formulations towards SARS-CoV-2 proteins for the potential treatment of COVID-19. In the current global emergency due to SARS-CoV-2 outbreak, passive immunotherapy emerges as a promising treatment for COVID-19. Among animal-derived products, equine formulations are still the cornerstone therapy for treating envenomations due to animal bites and stings. Therefore, drawing upon decades of experience in manufacturing snake antivenom, we developed and preclinically evaluated two anti-SARS-CoV-2 polyclonal equine formulations as potential alternative therapy for COVID-19. We immunized two groups of horses with either S1 (anti-S1) or a mixture of S1, N, and SEM mosaic (anti-Mix) ...
Development of a 3′:5′ digital PCR assay to determine horse mRNA integrity. Accurate tools to measure RNA integrity are essential to obtain reliable gene expression data. The reverse transcription quantitative PCR (RT-qPCR) based 3':5' assay permits a direct determination of messenger RNA (mRNA) integrity. However, the use of standard curves and the possible effect of PCR inhibitors make this method cumbersome and prone to variation, especially in small samples. Here we developed a triplex digital PCR (dPCR) 3':5' assay for assessing RNA integrity in equine samples as rapid and simple alternative to RT-qPCR. This dPCR assay not only provides a straight forward analysi...
Robustness of Digital PCR and Real-Time PCR in Transgene Detection for Gene-Doping Control. Gene doping is banned in human sports, horseracing, and equestrian sports. One possible form of gene doping is to administer exogenous genes, called transgenes. Several transgene detection methods based on quantitative PCR have been developed. In this study, we investigated the robustness of digital PCR and real-time PCR in transgene detection using primers and probes that matched (P-true) or incompletely matched (P-false) the template DNA. Fluorescence intensity was significantly reduced when substituted probes were used compared to that using the matched probe in both digital and real-time P...
Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase. Theileria equi, an intraerythrocytic protozoan parasite, causes equine piroplasmosis, a disease which negatively impacts the global horse industry. Genetic manipulation is one of the research tools under development as a control method for protozoan parasites, but this technique needs to be established for T. equi. Herein, we report on the first development of a stable transgenic T. equi line expressing enhanced green fluorescent protein/blasticidin S deaminase (eGFP/BSD). To express the exogenous fusion gene in T. equi, regulatory regions of the elongation factor-1 alpha (ef-1α) gene were id...
A Fast PCR Test for the Simultaneous Identification of Species and Gender in Horses, Donkeys, Mules and Hinnies. Having considered that the current methods are costly and time-consuming, we designed an only 3 pairs primer-based PCR test to accurately identify the species and gender in horses, donkeys, mules and hinnies. Through a thorough sequence comparison between horse and donkey's highly similar genomes, and a vast amount of preliminary confirmation, we found that three fragments, CNGB3 gene on an autosome, displacement loop region on mitochondrial DNA and SRY genes on chromosome Y, within these equine DNA, are enough to enable us achieving our goal. The PCR test described here would be an economical...
Internal cavity amplification of shell-like ferritin regulated with the change of the secondary and tertiary structure induced by PEF technology. In this study, the effect of pulsed electric field (PEF) on apparent morphology and molecular structure of shell-like ferritin obtained from horse spleen was determined by circular dichroic (CD), fluorescence spectroscopy, Raman spectroscopy, cold field emission scanning electron microscopy (CF-SEM) and transmission electron microscopy (TEM), and verified by molecule dynamics (MD) simulation. After PEF treatment, the α-helix content of the samples reached a minimum value at 10 kV/cm, which indicated that the ferritin structure has been partially unfolded. However, the α-helix content peaked ...
Hyaluronic Acid as Macromolecular Crowder in Equine Adipose-Derived Stem Cell Cultures. The use of macromolecular crowding in the development of extracellular matrix-rich cell-assembled tissue equivalents is continuously gaining pace in regenerative engineering. Despite the significant advancements in the field, the optimal macromolecular crowder still remains elusive. Herein, the physicochemical properties of different concentrations of different molecular weights hyaluronic acid (HA) and their influence on equine adipose-derived stem cell cultures were assessed. Within the different concentrations and molecular weight HAs, the 10 mg/mL 100 kDa and 500 kDa HAs exhibited the high...
Comparison between Allogenic and Xenogenic Bone Blocks on the Osteogenic Potential of Cultured Human Periodontal Ligament Stem Cells: Confocal Laser and Scanning Electron Microscopy Study. The aim of this study was to compare between equine and human bone blocks in the osteogenic differentiation of cultured human periodontal ligament stem cells (hPDLSCs) at 14 and 21 days of culture, using confocal laser microscopy and scanning electron microscopy. Methods: cultures of commercially obtained hPDLSCs were seeded onto equine and human bone blocks. At 14 days and 21 days of culture, confocal laser microscope images were obtained to assess cellular differentiation and adhesion, and scanning electron microscope images were obtained to validate the osteogenic differentiation by showin...
Antimicrobial Resistance and Extended-Spectrum Beta-Lactamase (ESBL) Genes in E. coli Isolated from Equine Fecal Samples in Turkey. The extensive use of antibacterial agents used for treatment in human and veterinary clinics to increase yield in livestock and aquaculture in developing countries causes the detection and spread of multidrug-resistant (MDR) strains in different sources. The presence of Escherichia coli strains is very common in racehorses and it's a serious problem on horse farms. Extended-spectrum beta-lactamase (ESBL) production is an important source of MDR development in Gram-negative strains such as E.coli. This study aimed to detect the presence of ESBL genes in E. coli strains isolated from horse farms...
Administration of pilocarpine by microneedle patch as a novel method for cystic fibrosis sweat testing. The sweat test is the gold standard for the diagnosis of cystic fibrosis (CF). The test utilizes iontophoresis to administer pilocarpine to the skin to induce sweating for measurement of chloride concentration in sweat. However, the sweat test procedure needs to be conducted in an accredited lab with dedicated instrumentation, and it can lead to inadequate sweat samples being collected in newborn babies and young children due to variable sweat production with pilocarpine iontophoresis. We tested the feasibility of using microneedle (MN) patches as an alternative to iontophoresis to administer ...
Zonal characterization and differential trilineage potentials of equine intrasynovial deep digital flexor tendon-derived cells. Intrasynovial deep digital flexor tendon (DDFT) injuries occur frequently and are often implicated in cases of navicular disease with poor outcomes and reinjuries. Cell-based approaches to tendon healing are gaining traction in veterinary medicine and ultimately may contribute to improved DDFT healing in horses. However, a better understanding of the innate cellular characteristics of equine DDFT is necessary for developing improved therapeutic strategies. Additionally, fibrocartilaginous, intrasynovial tendons like the DDFT are common sites of injury and share a poor prognosis across species,...
A Critical Overview of the Use of Platelet-Rich Plasma in Equine Medicine Over the Last Decade. In the 1990s, the role of platelets in inflammation and tissue healing was finally recognized. Since then, the clinical use of platelet-derived products (hemocomponents), such as, platelet-rich plasma (PRP), markedly increased. The promise of a more economical option of a disease-modifying treatment led to the intensive and continuous research of PRP products and to its widespread clinical use. A number of protocols and commercial kits have been developed with the intention of creating a more practical and reliable option for clinical use in equine patients. Still, the direct comparison betwee...
Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectroscopy for the Quantitative Analysis of Deuterium in Plasma: Application to Total Body Water Determination in Humans and Other Animals. Conventional methods for measuring the concentration of deuterium in body fluids are by either isotope ratio mass spectrometry or Fourier transform infrared transmission (FT-IR) spectroscopy. The latter method is often preferred as it is less expensive and time consuming; however, having a lower sensitivity means a larger sample volume is required. This study investigated an alternative FT-IR spectroscopic method, attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR), which has the potential to provide shorter analysis times while requiring smaller sample volumes. Deu...
An inactivated bacterium (paraprobiotic) expressing Bacillus thuringiensis Cry5B as a therapeutic for Ascaris and Parascaris spp. infections in large animals. and are important parasites in the family Ascarididae, large, ubiquitous intestinal-dwelling nematodes infecting all classes of vertebrates. Parasitic nematode drug resistance in veterinary medicine and drug recalcitrance in human medicine are increasing worldwide, with few if any new therapeutic classes on the horizon. Some of these parasites are zoonotic, , is passed from humans to pigs and . The development of new therapies against this family of parasites would have major implications for both human and livestock health. Here we tested the therapeutic ability of a paraprobiotic or dead ...
Development of Electrochemical DNA Biosensor for Equine Hindgut Acidosis Detection. The pH drop in the hindgut of the horse is caused by lactic acid-producing bacteria which are abundant when a horse's feeding regime is excessively carbohydrate rich. This drop in pH below six causes hindgut acidosis and may lead to laminitis. Lactic acid-producing bacteria and have been found to produce high amounts of L-lactate and D-lactate, respectively. Early detection of increased levels of these bacteria could allow the horse owner to tailor the horse's diet to avoid hindgut acidosis and subsequent laminitis. Therefore, 16s ribosomal ribonucleic acid (rRNA) sequences were identified a...
The inhibition of spermatic cystine/glutamate antiporter xCT (SLC7A11) influences the ability of cryopreserved stallion sperm to bind to heterologous zonae pellucidae. Sperm are redox-regulated cells, and deregulation of their redox status is considered to affect male fertility and to reduce their fertilizing ability following biotechnological procedures, such as cryopreservation. Cystine (CysS), after incorporation in sperm via SLC7A11 antiporter, has been demonstrated to increase intracellular GSH content, the most important non enzymatic antioxidant. This study was aimed at investigating the role of SLC7A11 antiporter on frozen-thawed stallion sperm ability to respond to in vitro capacitating environment after post-thaw incubation with CysS and/or Sulfas...
Validation of a flow cytometric assay to detect intraerythrocytic reactive oxygen species in horses. Oxidative stress refers to the accumulation of reactive oxygen species (ROS). Most assays for ROS detection are costly, laborious, and usually use indirect markers. The use of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) is a possible alternative. This substance becomes a fluorochrome when oxidized by ROS, with the resultant fluorescence proportional to ROS concentration. Erythrocytes are highly exposed to ROS, resulting in cell damage and consequently impaired oxygen delivery. The effects of this exposure in physiologic and pathologic conditions necessitate an improvement in ROS detec...