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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.
Establishment of cloned Anaplasma phagocytophilum and analysis of p44 gene conversion within an infected horse and infected SCID mice. Diverse p44 alleles at the p44 expression locus (p44Es) encoding surface-exposed major membrane proteins, P44s, of Anaplasma phagocytophilum were hypothesized to be garnered by recombination to enact antigenic variation. However, this hypothesis has not been proven so far, due to inability to clone this obligate intragranulocytic rickettsia. To define the p44E recombination, we developed a novel method to clone A. phagocytophilum. This isogenic cloned population containing a defined p44E was used to infect a naive horse and severe combined immunodeficiency (SCID) mice. During a 58-day infectio...
Sequence analysis of canine and equine ferritin H and L subunit cDNAs. Canine and equine ferritin H and L subunit cDNA clones were obtained using reverse transcriptase-polymerase chain reaction (RT-PCR) and TA cloning from various tissues. Canine liver and spleen ferritin H subunit cDNA clones contained an open reading frame for the same 182-amino acid protein as that reported in canine brain ferritin H subunit cDNA although there were substitutions in the 3'-noncoding regions. Ferritin L subunit cDNA clones from canine liver, spleen, and kidney showed identical coding sequences encoding the 174-amino acid protein except for a single nucleotide substitution in ki...
Detection of differentially regulated genes in ischaemic equine intestinal mucosa. Colic is a serious disease syndrome in horses. Much of the mortality is associated with ischaemic-injured intestine during strangulating obstruction, yet there is limited understanding of the associated molecular events. Identification of differentially expressed genes during ischaemic injury should expand our understanding of colic and may lead to novel targeted therapeutic approaches in the future. Objective: To isolate and identify differentially expressed genes in equine jejunum following a 2 h ischaemic event compared to normally perfused jejunum. Methods: Suppressive subtractive hybridis...
Structure of myelin P2 protein from equine spinal cord. Equine P2 protein has been isolated from horse spinal cord and its structure determined to 2.1 A. Since equine myelin is a viable alternative to bovine tissue for large-scale preparations, characterization of the proteins from equine spinal cord myelin has been initiated. There is an unusually high amount of P2 protein in equine CNS myelin compared with other species. The structure was determined by molecular replacement and subsequently refined to an R value of 0.187 (Rfree=0.233). The structure contains a molecule of the detergent LDAO and HEPES buffer in the binding cavity and is otherwise ...
The development and application of the modern reproductive technologies to horse breeding. Although the horse was probably the first animal to experience and benefit from artificial insemination, it trailed the field somewhat with regard to the application of embryo transfer and other oocyte and embryo-related modern breeding technologies. But with a late run it is now back in mid-field and gaining fast on the other large domestic species in the application of the many technological advances of the past 20 years to sound breeding practice. Improvements in extenders and cryoprotectants have resulted in a veritable upsurge in the transport and insemination of cooled and frozen stallio...
Ocular parameters related to drug delivery in the canine and equine eye: aqueous and vitreous humor volume and scleral surface area and thickness. To measure the ocular surface area, scleral thickness, and the aqueous and vitreous humor volumes in the canine and equine eye. Animals studied Fourteen canine and 16 equine cadaver eyes. Methods: Eyes were measured either fresh or following fixation in formalin. Ocular surface area was calculated using the fluid displacement method. Globes were hemisected and aqueous and vitreous humor were collected and quantitated. Scleral thickness was measured by digital caliper, by image projection, and by ultrasonic biomicroscopy (UBM). Results: Mean +/- standard deviation (SD) scleral surface areas in ...
In vitro evaluation of the contractile response to endothelin-1 of the circular and longitudinal myometrial layers of the uterine horn of nongravid mares. To characterize the in vitro response of circular and longitudinal myometrial layers of the uterine horn (CMLH and LMLH, respectively) of horses to endothelin (ET)-1 by use of specific ETA (BQ-123) and ETB (IRL-1038) receptor antagonists. Methods: Uteruses from 10 nongravid mares in anestrus. Methods: Muscle strips from the CMLH and LMLH were suspended in tissue baths and connected to force-displacement transducers interfaced with a polygraph. Strips were incubated for 45-minute intervals with no antagonist (control specimens), and 3 concentrations (10(-9), 10(-7), and 10(-5)M) of BQ-123, IRL-...
Update on equine ICSI and cloning. Intracytoplasmic sperm injection (ICSI) has recently become efficient enough to be considered for clinical use. With ICSI, one spermatozoa is injected into a mature oocyte. Harvesting of an oocyte ex vivo, followed by ICSI and transfer of the fertilized oocyte to the oviduct, may be applicable when semen quality is insufficient for standard insemination. Sperm injection, followed by in vitro embryo culture to the blastocyst stage, may be used in cases where multiple oocytes are to be fertilized (e.g. when oocytes are collected post-mortem). Nuclear transfer (cloning) of horses is possible but ...
Immunologic testing of xeno-derived osteochondral grafts using peripheral blood mononuclear cells from healthy human donors. One means of treating osteoarthritis is with autologous or allogeneic osteochondral grafts. The purpose of this study was to evaluate the innate immunological response in humans toward xeno-derived osteochondral grafts that have been partially or entirely treated by the photooxidation process. Methods: The antigens tested included bovine, porcine, ovine and equine osteochondral samples that have been treated in successive steps of photooxidation. ELISPOT assays were used to evaluate the production of IL-1, IL-4, IL-6, IL-10, IL-12 and TNF-alpha by human monocytes in response to the antigens. R...
A tumor necrosis factor receptor family protein serves as a cellular receptor for the macrophage-tropic equine lentivirus. Characterization of cellular receptors for human, simian, and feline immunodeficiency viruses that are tropic for lymphocytes and macrophages have revealed a common theme of a sequential binding of viral envelope proteins with two coreceptors to mediate virus infection of target cells. In contrast to these dual tropic immunodeficiency viruses, the ungulate lentiviruses, including equine infectious anemia virus (EIAV), exclusively infect cells of the monocyte-macrophage lineage to cause progressive degenerative diseases without clinical immunodeficiency. EIAV causes a uniquely dynamic disease t...
Comparison of cell proliferation index in equine and caprine embryos using a modified BrdU incorporation assay. The measurement of cell proliferation and cell viability using 5'bromo-2'deoxy-uridine (BrdU) labelling has been described in several cell types and species. The aim of this study was to adapt this technique to equine embryos and to compare the index of DNA replication (S-phase) between equine and caprine embryos. Seventeen equine embryos were recovered at day 6.5 post-ovulation and 20 caprine embryos were recovered at day 7 after the onset of estrus. Equine embryos were incubated during 1h at 39 degrees C in PBS containing 1mM of BrdU. Embryos were then treated in 0.05% trypsin during 15 min ...
Kerr-gated time-resolved Raman spectroscopy of equine cortical bone tissue. Picosecond time-resolved Raman spectroscopy in equine cortical bone tissue is demonstrated. Using 400-nm pulsed laser excitation (1 ps at 1 kHz) it is shown that Kerr cell gating with a 4-ps window provides simultaneously time-resolved rejection of fluorescence and time-resolved Raman scatter enabling depth profiling through tissue. The Raman shifts are the same as those observed by conventional cw Raman spectroscopy using deep-red or near-infrared lasers. The time decay of Raman photons is shown to fit an inverse square root of time function, suggesting propagation by a diffusive mechanism. U...
Practical uses for ecdysteroids in mammals including humans: an update. Ecdysteroids are widely used as inducers for gene-switch systems based on insect ecdysteroid receptors and genes of interest placed under the control of ecdysteroid-response elements. We review here these systems, which are currently mainly used in vitro with cultured cells in order to analyse the role of a wide array of genes, but which are expected to represent the basis for future gene therapy strategies. Such developments raise several questions, which are addressed in detail. First, the metabolic fate of ecdysteroids in mammals, including humans, is only poorly known, and the rapid catabo...
Potential of equine herpesvirus 1 as a vector for immunization. Key problems using viral vectors for vaccination and gene therapy are antivector immunity, low transduction efficiencies, acute toxicity, and limited capacity to package foreign genetic information. It could be demonstrated that animal and human cells were efficiently transduced with equine herpesvirus 1 (EHV-1) reconstituted from viral DNA maintained and manipulated in Escherichia coli. Between 13 and 23% of primary human CD3+, CD4+, CD8+, CD11b+, and CD19+ cells and more than 70% of CD4+ MT4 cells or various human tumor cell lines (MeWo, Huh7, HeLa, 293T, or H1299) could be transduced with o...
Expression of equine glucose transporter type 4 in skeletal muscle after glycogen-depleting exercise. To clone and sequence cDNA for equine insulin-responsive glucose transporter (glucose transporter type 4 [GLUT-4]) and determine effects of glycogen-depleting exercise and meal type after exercise on GLUT-4 gene expression in skeletal muscle of horses. Methods: Muscle biopsy specimens from 7 healthy adult horses. Methods: Total RNA was extracted from specimens, and GLUT-4 cDNA was synthesized and sequenced. Horses were exercised on 3 consecutive days. On the third day of exercise, for 8 hours after exercise, horses were either not fed, fed half of daily energy requirements as hay, or fed an is...
A recombinant envelope protein vaccine against West Nile virus. West Nile (WN) virus is a flavivirus that first appeared in North America in 1999. Since then, more than 600 human deaths and 22,000 equine infections have been attributed to the virus in the United States. We expressed a truncated form of WN virus envelope (E) protein in Drosophila S2 cells. This soluble recombinant E protein was recognized by antibodies from naturally infected horses, indicating that it contains native epitopes. Mice and horses produced high-titer antibodies when immunized with recombinant E protein combined with aluminum hydroxide. Immunized mice were resistant to challenge...
Quantitative analysis of voltage-gated potassium currents from primary equine (Equus caballus) and elephant (Loxodonta africana) articular chondrocytes. In this comparative study, we have established in vitro models of equine and elephant articular chondrocytes, examined their basic morphology, and characterized the biophysical properties of their primary voltage-gated potassium channel (Kv) currents. Using whole cell patch-clamp electrophysiological recording from first-expansion and first-passage cells, we measured a maximum Kv conductance of 0.15 +/- 0.04 pS/pF (n = 10) in equine chondrocytes, whereas that in elephant chondrocytes was significantly larger (0.8 +/- 0.4 pS/pF, n = 4, P </= 0.05). Steady-state activation parameters of eleph...
Horse cytokine/IgG fusion proteins–mammalian expression of biologically active cytokines and a system to verify antibody specificity to equine cytokines. Recombinant cytokines are valuable tools for functional studies and candidates for vaccine additives or therapeutic use in various diseases. They can also be used to generate specific antibodies to analyze the roles of different cytokines during immune responses. We generated a mammalian expression system for recombinant cytokines using the equine IgG1 heavy chain constant region as a tag for detection and purification of the expressed cytokine, demonstrated here using equine interferon-gamma (IFN-gamma), interleukin-2 (IL-2), interleukin-4 (IL4) and transforming growth factor-beta1 (TGF-beta1...
Polymorphisms of the upstream regulatory region of the major histocompatibility complex DRB genes in domestic horses. Sequence information was obtained on the variation of the ELA-DRB upstream regulatory region (URR) after polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) cloning and sequencing of approximately 220 bp upstream of the first exon of horse DRB genes. The sequence of the proximal URR of equine DRB is composed of highly conserved sequence motifs, showing the presence of the W, X, Y, CAAT and TATA conserved boxes of major histocompatibility complex (MHC) class II promoters. Five different polymorphic horse DRB promoter sequences were detected in five horse breeds. The res...
In vivo evaluation of an EIAV vector for the systemic genetic delivery of therapeutic antibodies. Lentiviral-based vectors hold great promise as gene delivery vehicles for the treatment of a wide variety of diseases. We have previously reported the development of a nonprimate lentiviral vector system based on the equine infectious anaemia virus (EIAV), which is able to efficiently transduce dividing and nondividing cells both in vitro and in vivo. Here, we report on the application of EIAV vectors for the systemic delivery of an antibody fusion protein designed for the treatment of cancer. The therapeutic potential of a single chain antibody against the tumour-associated antigen, 5T4, fuse...
Cloning and expression of equine insulin-like growth factor binding proteins in normal equine tendon. To define a portion of the nucleotide sequences of each of the 6 insulin-like growth factor (IGF) binding proteins (IGFBPs) in horses and describe patterns of messenger RNA (mRNA) and protein expression for IGFBPs in normal equine tendons. Methods: 7 horses. Methods: Total RNA was extracted from the tensile region of normal superficial digital flexor tendons and reverse transcribed into complimentary DNA (cDNA). The cDNA was amplified via PCR, and products representing portions of each IGFBP were cloned and sequenced. Nucleotide sequences were used to deduce the amino acid sequences, and both ...
Cloning and expression of type III collagen in normal and injured tendons of horses. To clone the 5' end of type III collagen and describe its pattern of mRNA and protein expression in normal and healing tendons in horses. Methods: 14 healthy adult horses. Methods: The tensile region of collagenase-injured superficial digital flexor tendons was harvested at intervals from 1 to 24 weeks after injury. Total RNA was reverse-transcribed into cDNA for cloning and sequencing of type III collagen. Equine-specific nucleic acid probes were developed and used for northern blot analysis and in situ hybridization. Type III collagen protein and cyanogen bromide-cleaved collagen peptides we...
On the difference in stability between horse and sperm whale myoglobins. The work in the literature on apomyoglobin is almost equally divided between horse and sperm whale myoglobins. The two proteins share high homology, show similar folding behavior, and it is often assumed that all folding phenomena found with one protein will also be found with the other. We report data at equilibrium showing that horse myoglobin was 2.1 kcal/mol less stable than sperm whale myoglobin at pH 5.0, and aggregated at high concentrations as measured by gel filtration and analytical ultracentrifugation experiments. The higher stability of sperm whale myoglobin was identified for both...
GPX5 orthologs of the mouse epididymis-restricted and sperm-bound selenium-independent glutathione peroxidase are not expressed with the same quantitative and spatial characteristics in large domestic animals. We report here on the cloning of cDNAs coding bovine and equine orthologs of mouse epididymis-restricted and sperm-bound glutathione peroxidase 5 (GPX5), a selenium-independent member of the multigenic GPX family in mammals. The complete sequence of bovine GPX5 as well as a partial sequence of the equine GPX5 were characterized, conceptually translated and aligned with other known mammalian GPX5 proteins. Using Northern blotting assays, we show that the level of expression of GPX5 is high in bovine but low in equine and that in both species the regionalization of GPX5 expression in epididymis ...
Cloned horse pregnancies produced using adult cumulus cells. The objectives of the present study were to: (1) clone horses using adult cumulus cells; and (2) determine whether the cumulus cell donor affected the outcome. In vivo-matured cumulus-oocyte complexes were obtained using transvaginal ultrasound-guided follicle aspiration; oocytes were used as cytoplasts, whereas cumulus cells (from one of three different mares) were used as donor cells. Immediately following nuclear transfer and activation procedures, cloned embryos were transferred surgically to the oviduct of recipient mares (n = 2-5 embryos per recipient) that had ovulated within 24 h prior...
High pressure flow cytometric sorting damages sperm. Sexing sperm by high-speed flow cytometry subjects them to high pressure. The routine operating pressure of the MoFlo SX flow cytometer for sperm sorting for commercial production has been 50 pounds/square inch (psi), with a standard 70 microm standard nozzle tip. It was hypothesized that lowering the sorting pressure could reduce sperm damage. Therefore, a series of experiments using semen from six bulls, sorted with three MoFlo SX sorters, was conducted to determine optimal pressure. An additional experiment was done with stallion spermatozoa. In Experiment 1, sorting at 30 psi compared to 5...
The improvement of the therapeutic anti-Lachesis muta serum production in horses. The main features associated with pit viper envenomations include the intense local lesions such as oedema, necrosis, acute renal failure and other effects. The severity of these reactions to snakebite depends on the degree of envenomation. Lachesis muta venom (LMV) has weak lethal activity, but due to the large amount often inoculated, the effects are extremely severe and demand anti-venom with a high neutralizing capacity. LMV had the lowest neutralizing antibody induction capacity in horses when compared with that of other venoms. For example, Bothrops anti-venom serum neutralizes 180 times...
