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.
Isolation of endothelial colony-forming cells from blood samples collected from the jugular and cephalic veins of healthy adult horses. OBJECTIVE To evaluate optimal isolation of endothelial colony-forming cells (ECFCs) from peripheral blood of horses. SAMPLE Jugular and cephalic venous blood samples from 17 adult horses. PROCEDURES Each blood sample was divided; isolation was performed with whole blood adherence (WBA) and density gradient centrifugation (DGC). Isolated cells were characterized by uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL), vascular tubule formation, and expression of endothelial (CD34, CD105, vascular endothelial growth ...
Beta-mercaptoethanol supplementation of in vitro maturation medium does not influence nuclear and cytoplasmic maturation of equine oocytes. In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low-molecular thiol compounds can be added to culture media. Beta-mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1 mM) and high (0.7 mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recove...
Detection of single nucleotide polymorphisms (SNP) in equine coat color genes using SNaPshotTM multiplex kit or pluronic F-108 tri-block copolymer and capillary electrophoresis. Molecular methods for the detection of mammalian coat color phenotypes have expanded greatly within the past decade. Many phenotypes are associated with a single nucleotide polymorphism mutation in the genetic sequence. Traditionally, these mutations are detected through sequencing, hybridization assays or mini-sequencing. However, these techniques can be expensive and tedious. Previously, CE-SSCP using the F-108 polymer was able to distinguish SNPs for the melanocortin-1 receptor (mc1r) coat color gene in horses (Equus caballus) that differed by one nucleotide substitution. The objective of t...
Chiral Selectivity in Inter-reactant Recognition and Electron Transfer of the Oxidation of Horse Heart Cytochrome c by Trioxalatocobaltate(III). Outer-sphere electron transfer (ET) between optically active transition-metal complexes and either other transition-metal complexes or metalloproteins is a prototype reaction for kinetic chirality. Chirality as the ratio between bimolecular rate constants of two enantiomers mostly amounts to 1.05-1.2 with either the Λ or Δ form the more reactive, but the origin of chirality in ET parameters such as work terms, electronic transmission coefficient, and nuclear reorganization free energy has not been addressed. We report a study of ET between the Λ-/Δ-[Co(Ox)3](3-) pair (Ox = oxalate) and hor...
A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase. Equine type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense mutation (R309H) in the glycogen synthase (GYS1) gene, enhanced glycogen synthase (GS) activity and excessive glycogen and amylopectate inclusions in muscle. Equine muscle biochemical and recombinant enzyme kinetic assays in vitro and homology modelling in silico, were used to investigate the hypothesis that higher GS activity in affected horse muscle is caused by higher GS expression, dysregulation, or constitutive activation via a conformational change. PSSM1-affected horse muscle had significantly higher gly...
Amino acid mutations in the env gp90 protein that modify N-linked glycosylation of the Chinese EIAV vaccine strain enhance resistance to neutralizing antibodies. The Chinese EIAV vaccine is an attenuated live virus vaccine obtained by serial passage of a virulent horse isolate (EIAV) in donkeys (EIAV) and, subsequently, in donkey cells in vitro. In this study, we compare the env gene of the original horse virulent virus (EIAV) with attenuated strains serially passaged in donkey MDM (EIAV) and donkey dermal cells (EIAV). Genetic comparisons among parental and attenuated strains found that vaccine strains contained amino acid substitutions/deletions in gp90 that resulted in a loss of three potential N-linked glycosylation sites, designated g5, g9, and g1...
Reproductive stage-dependent effects of additional cryoprotectant agents for the cryopreservation of stallion germ cells. The main objective of this study was to evaluate the efficacy of an additional cryoprotectant in 10% dimethyl sulfoxide (DMSO) on cryopreserving germ cells from stallions at different reproductive stages. Testicular samples were obtained from pre-pubertal (1-1.5 yr, n=6) and post-pubertal (3-7 yr, n=5) stallions. Germ cells were isolated using a two-enzyme digestion procedure and cryopreserved in minimal essential medium alpha containing 10% fetal bovine serum and 10% DMSO with or without addition of trehalose (50, 100, or 200mM) or polyethylene glycol (PEG, 2.5, 5, or 10%). Viability, cell po...
Bone morphogenetic protein 2 stimulates chondrogenesis of equine synovial membrane-derived progenitor cells. Bone morphogenetic protein 2 (BMP-2) is critical for skeletal and cartilage development, homeostasis and repair. This study was conducted to clone and characterize equine BMP-2, develop expression constructs for equine BMP-2, and to determine whether BMP-2 can stimulate chondrogenesis of equine synovial membrane-derived progenitor cells (SMPC). Methods: Equine BMP-2 cDNA was amplified from chondrocyte RNA, and then transferred into an expression plasmid and adenoviral vector. Effective expression of equine BMP-2 was confirmed using a BMP reporter cell line. SMPC were isolated from synovium, ex...
Bone structural similarity score: a multiparametric tool to match properties of biomimetic bone substitutes with their target tissues. One of the hardest tasks in developing or selecting grafts for bone substitution surgery or tissue engineering is to match the structural and mechanical properties of tissue at the recipient site, because of the large variability of tissue properties with anatomical site, sex, age and health conditions of the patient undergoing implantation. We investigated the feasibility of defining a quantitative bone structural similarity score based on differences in the structural properties of synthetic grafts and bone tissue. Methods: Two biocompatible hydroxyapatite porous scaffolds with different nom...
Synthesis and evaluation of isometamidium-alginate nanoparticles on equine mononuclear and red blood cells. Isometamidium hydrochloride (ISMM) is an effective drug for the treatment of trypanosomosis, but it causes local and systemic toxicity. Isometamidium hydrochloride has limited therapeutic index and exhibit considerable variation in their prophylactic activities. We developed a trypanocidal nanoformulation using ISMM and polymers sodium alginate/gum acacia to enhance the efficacy of the drug at lower doses, while minimizing undesirable side effects. It was characterized by transmission electron microscopy and infrared spectroscopy for evaluation of size, morphology, functional groups, etc. In v...
Comparison of a modified phenol/chloroform and commercial-kit methods for extracting DNA from horse fecal material. There are many choices for methods of extracting bacterial DNA for Next Generation Sequencing (NGS) from fecal samples. Here, we compare our modifications of a phenol/chloroform extraction method plus an inhibitor removal solution (C3) (ph/Chl+C3) to the PowerFecal® DNA Isolation Kit (MoBio-K). DNA quality and quantity coupled to NGS results were used to assess differences in relative abundance, Shannon diversity index, unique species, and principle coordinate analysis (PCoA) between biological replicates. Six replicate samples, taken from a single ball of horse feces manually collected from ...
Establishment of an equine tetanus antitoxin reference standard for veterinary use in Japan. To establish the first National Veterinary Assay Laboratory (NVAL) equine tetanus antitoxin reference standard for veterinary use, we manufactured vials of a candidate antitoxin. These were quality tested for moisture content, vacuum, colour, clarity, and the presence of foreign objects. Ultimately, 115 quality-controlled vials were prepared. To estimate the antitoxin potency of the candidate standard, three different laboratories conducted parallel line assays alongside the existing antitoxin standard. These potency estimates ranged from 38 to 42 IU. This activity was maintained for two years...
Genetic testing in veterinary dermatology. Molecular genetics has made significant advances in the analysis of hereditary dermatoses during the last several years. Objective: To provide an update on currently available genetic tests for skin diseases of dogs, cats and horses, and to aid the veterinary clinician in the appropriate selection and applications of genetic tests. Methods: The scientific literature on the topic was critically reviewed. The list of known causative variants for genodermatoses and hair morphology traits was compiled by searching the Online Mendelian Inheritance in Animals (OMIA) database. Results: Genetic testin...
A concise review of common animal models for the study of limb regeneration. Correct selection of an appropriate animal mode to closely mimic human extremity diseases or to exhibit desirable phenotypes of limb regeneration is the first critical step for all scientists in biomedical and regenerative researches. The commonly-used animals in limb regeneration and repairing studies, such as axolotl, mice, and rats, are discussed in the review and other models including cockroaches, dogs, and horses are also mentioned. The review weighs the general advantages, disadvantages, and precedent uses of each model in the context of limb and peripheral injury and subsequent regener...
A new strategy for the decellularisation of large equine tendons as biocompatible tendon substitutes. Tendon ruptures and/or large losses remain to be a great clinical challenge and often require full replacement of the damaged tissue. The use of auto- and allografts or engineered scaffolds is an established approach to restore severe tendon injuries. However, these grafts are commonly related to scarce biocompatibility, site morbidity, chronic inflammation and poor biomechanical properties. Recently, the decellularisation techniques of allo- or xenografts using specific detergents have been studied and have been found to generate biocompatible substitutes that resemble the native tissue. This...
Long-Term Cell Tracking Following Local Injection of Mesenchymal Stromal Cells in the Equine Model of Induced Tendon Disease. Tendon disease has been treated with multipotent mesenchymal stromal cells (MSCs) in the equine large-animal model with promising success. The aim of this study was to gain more insight into the fate and biodistribution of MSCs after local application into tendon lesions by long-term cell tracking in this large-animal model. Superficial digital flexor tendon lesions were induced in all limbs in six horses and injected with 10106 Molday ION Rhodamine B-labeled MSCs suspended in serum or serum alone. Follow-up was performed using low-field magnetic resonance imaging (MRI), flow cytometry, and hi...
In vitro effects of platelet-rich gel supernatants on histology and chondrocyte apoptosis scores, hyaluronan release and gene expression of equine cartilage explants challenged with lipopolysaccharide. Platelet-rich plasma (PRP) preparations are a common treatment in equine osteoarthritis (OA). However, there are controversies regarding the ideal concentration of platelets and leukocytes in these biological substances necessary to induce an adequate anti-inflammatory and anabolic response in articular cartilage. The aims were to study the influence of leukocyte- and platelet-rich gel (L-PRG) and pure platelet-rich gel (P-PRG) supernatants on the histological changes of cartilage, the degree of chondrocyte apoptosis, the production of hyaluronan (HA) and the gene expression of nuclear factor ...
Destabilase-lysozyme-2 – original recombinant thrombolytic preparation of medicinal leech inhibits horse platelets aggregation. The purpose. Identifying the capacity of the medicinal leech novel original recombinant thrombolytic preparation Destabilase-Lysozyme-2 to inhibit the blood platelet aggregation. Gene of destabilase-lysozyme. ds2 (mlDL-Ds2 ), was cloned in E.coli cells. Recombinant protein was isolated in denaturing conditions using metal-chelate chromatography followed by denaturation of the polypeptide by rapid dilution in exact accordance with the procedure described by Kurdyumov A.S. et al. ( 2016, Russian Journal of Bioorganic Chemistry, v.42, s. 42-52). Blood was collected from the jugular vein of 18 hor...
Optimized methods for extracting circulating small RNAs from long-term stored equine samples. Circulating miRNAs in body fluids, particularly serum, are promising candidates for future routine biomarker profiling in various pathologic conditions in human and veterinary medicine. However, reliable standardized methods for miRNA extraction from equine serum and fresh or archived whole blood are sorely lacking. We systematically compared various miRNA extraction methods from serum and whole blood after short and long-term storage without addition of RNA stabilizing additives prior to freezing. Time of storage at room temperature prior to freezing did not affect miRNA quality in serum. Fur...
Development of a chicken-derived antivenom against the taipan snake (Oxyuranus scutellatus) venom and comparison with an equine antivenom. A chicken-derived antivenom (ChDAv) towards taipan snake (Oxyuranus scutellatus) venom was produced by purifying anti-taipan IgY from egg yolks of hens immunized with taipan venom. The productivity, antivenomic profile, neutralization ability, pharmacokinetic properties and immunogenicity of the ChDAv were compared with those of an antivenom produced in horses (EDAv). We found that 382 eggs are required to produce the mass of anti-taipan antibodies contained in one liter of equine hyperimmune plasma, and that 63 chickens would be needed to generate the amount of anti-taipan antibodies annually...
Engineered Mesenchymal Cells Improve Passive Immune Protection Against Lethal Venezuelan Equine Encephalitis Virus Exposure. : Mesenchymal stromal cells (MSCs) are being exploited as gene delivery vectors for various disease and injury therapies. We provide proof-of-concept that engineered MSCs can provide a useful, effective platform for protection against infectious disease. Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne pathogen affecting humans and equines and can be used in bio-warfare. No licensed vaccine or antiviral agent currently exists to combat VEEV infection in humans. Direct antibody administration (passive immunity) is an effective, but short-lived, method of providing immediate prote...
Sternal bone marrow derived equine multipotent mesenchymal stromal cells (MSCs): investigations considering the sampling site and the use of different culture media. Aspiration of equine sternal bone marrow is required for the cultivation of bone marrow-derived multipotent mesenchymal stromal cells (BM-MSCs) for regenerative therapies. For bone marrow aspiration as well as for MSC cultivation, there is a need to optimize techniques and protocols to enhance MSC harvest at minimized culture times. In a comparative study bone marrow aspirates from sternebra 4 and 5 were collected at two different positions within the sternebrae, either from 10 mm or from 30 mm dorsal from the ventral margin of the sternebrae. Accuracy of the puncture depth was confirmed by ...
Cloning and expression of the 4D8 gene from Hyalomma asiaticum tick. Hyalomma asiaticum tick, an important ectozoic parasite causes tickle, pain, anemia, weight loss, and paralysis in its hosts, which include humans, cattle, sheep, horses, camels, and hares. The 4D8 gene can be a potential vaccine candidate antigen for H. asiaticum. In the present study, we cloned and expressed the 4D8 gene of H. asiaticum from Xinjiang Province. Primers were designed according to the H. asiaticum tick 4D8 gene sequence available in GenBank. The gene was amplified by reverse transcription-polymerase chain reaction and the fragments were subcloned into the prokaryotic expression...
Inflammation-induced transgene expression in genetically engineered equine mesenchymal stem cells. Osteoarthritis, a chronic and progressive degenerative joint disorder, ranks amongst the top five causes of disability. Given the high incidence, associated socioeconomic costs and the absence of effective disease-modifying therapies of osteoarthritis, cell-based treatments offer a promising new approach. Owing to their paracrine, differentiation and self-renewal abilities, mesenchymal stem cells (MSCs) have great potential for regenerative medicine, which might be further enhanced by targeted gene therapy. Hence, the development of systems allowing transgene expression, particularly when regu...
Effects of Hypoxia and Chitosan on Equine Umbilical Cord-Derived Mesenchymal Stem Cells. Chitosan opens new perspectives in regenerative medicine as it enhances the properties of mesenchymal stem cells (MSCs) through formation of spheroids. Hypoxia has also been proposed to enhance stemness and survival of MSCs after in vivo implantation. These characteristics are relevant to the development of an off-the-shelf source of allogenic cells for regenerative therapy of tendinopathies. Umbilical cord-derived MSCs (UCM-MSCs) offer an abundant source of immature and immunoprivileged stem cells. In this study, equine UCM-MSCs (eqUCM-MSCs) conditioned for 3 and 7 days on chitosan films at 5...
Fascicles and the interfascicular matrix show adaptation for fatigue resistance in energy storing tendons. Tendon is composed of rope-like fascicles, bound together by interfascicular matrix (IFM). Our previous work shows that the IFM is critical for tendon function, facilitating sliding between fascicles to allow tendons to stretch. This function is particularly important in energy storing tendons, which experience extremely high strains during exercise, and therefore require the capacity for considerable inter-fascicular sliding and recoil. This capacity is not required in positional tendons. Whilst we have previously described the quasi-static properties of the IFM, the fatigue resistance of the...
Scintigraphic Tracking of Allogeneic Mesenchymal Stem Cells in the Distal Limb After Intra-Arterial Injection in Standing Horses. To assess the feasibility of intra-arterial administration of allogeneic mesenchymal stem cells (MSC) in the median artery of standing horses and evaluate the distribution and retention of radiolabeled cells. Methods: In vivo experimental study. Methods: Six research horses. Methods: Technetium(99m) -HexaMethyl-Propylene-Amine Oxime-labeled MSC were injected under ultrasound guidance in the median artery of 6 front limbs of 3 horses, standing under sedation. Scintigraphic images were obtained at the time of injection, and at 1, 6, and 24 hours postinjection. Six additional limbs from 3 horses ...
Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity. An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI) promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV) in horse cells. In our study, the horse RNA PolI promoter was cloned from fetal equine lung cells. Using the luciferase assay, ...
Generation of Equine-Induced Pluripotent Stem Cells and Analysis of Their Therapeutic Potential for Muscle Injuries. Horse health has become a major concern with the expansion of horse-related industries and sports; the importance of healthy muscles for horse performance and daily activities is undisputed. Here we generated equine-induced pluripotent stem cells (E-iPSCs) by reprogramming equine adipose-derived stem cells (E-ADSCs) into iPSCs using a polycistronic lentiviral vector encoding four transcription factors (i.e., Oct4, Sox2, Klf4, and c-Myc) and then examined their pluripotent characteristics. Subsequently, established E-iPSCs were transplanted into muscle-injured Rag/ mdx mice. The histopathology ...