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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.
Characterization of a cytolytic strain of equine infectious anemia virus. A novel strain of equine infectious anemia virus (EIAV) called vMA-1c that rapidly and specifically killed infected equine fibroblasts (ED cells) but not other infectible cell lines was established. This strain was generated from an avirulent, noncytopathic strain of EIAV, MA-1. Studies with this new cytolytic strain of virus have permitted us to define viral parameters associated with EIAV-induced cell killing and begin to explore the mechanism. vMA-1c infection resulted in induction of rapid cell death, enhanced fusogenic activity, and increased rates of spread in equine fibroblasts compared...
Intracytoplasmic sperm injection of bovine oocytes with stallion spermatozoa. Five experiments were designed to study the fertilizability and development of bovine oocytes fertilized by intracytoplasmic sperm injection (ICSI) with stallion spermatozoa. Experiment 1 determined the time required for pronuclear formation after ICSI. Equine sperm head decondensation began 3 h after ICSI; 42% were decondensed 6 h after ICSI. Male pronuclei (MPN) began to form 12 h after ICSI. Female pronuclei (FPN), however, formed as early as 6 h after ICSI. In Experiment 2, ionomycin, ionomycin plus 6-dimethylaminopurine (DMAP), and thimerosal were used to activate ICSI ova. None of the IC...
Surfactant proteins in bronchoalveolar lavage fluid of horses: assay technique and changes following road transport. An enzyme-linked immunosorbent assay (ELISA) was developed for equine surfactant proteins SP-A and SP-D in bronchoalveolar lavage fluid (BALF). Anti-equine SP-A or SP-D monoclonal antibodies (mAb) were produced by hybridoma technology, purified by the antibody purification reagent, and analysed by Western blotting analysis. The immunoreaction (two-site sandwich ELISA) with a mAb, peroxidase-labelled mAb and BALF sample was carried out simultaneously and analytical recovery and precision were assayed. Six mAb for SP-A and four mAb for SP-D were successfully cloned in limiting dilution to monocl...
The development of a competitive PCR-ELISA for the detection of equine herpesvirus-1. Equine herpesvirus-1 (EHV-1) infection is of significant animal welfare and economic importance. Yet, no standardised molecular techniques are available for diagnosis or confirmation of viral infection. The purpose of this study was to develop a standardised and quantitative assay system for the reliable detection of EHV-1 infection which was capable of eliminating the likelihood of false negative results. A region within the EHV-1 glycoprotein B gene was amplified by polymerase chain reaction (PCR), cloned and subjected to site-directed mutagenesis to generate a control plasmid, amplifiable b...
The use of laser microdissection for the preparation of chromosome-specific painting probes in farm animals. Laser microbeam microdissection and laser pressure catapulting procedure were used for the construction of chromosome-specific painting probes, arm-specific probes and probes for chromosomal subfragments. We report on a method for generation of fluorescence in-situ hybridization probes from laser dissected chromosomes of farm animals. So far, using the described method, a set of chromosome-specific painting probes has been obtained for all porcine chromosomes, 17 chromosomes of cattle and selected equine chromosomes. It is concluded that the laser technology appears to be a useful and powerful...
Embryo technologies in the horse. Recent studies demonstrated that zwitterionic buffers could be used for satisfactory storage of equine embryos at 5 degrees C. The success of freezing embryos is dependent upon size and stage of development. Morulae and blastocysts <300 microm can be slowly cooled or vitrified with acceptable pregnancy rates after transfer. The majority of equine embryos are collected from single ovulating mares, as there is no commercially available product for superovulation in equine. However, pituitary extract, rich in FSH, can be used to increase embryo recovery three- to four-fold. Similar to human medic...
New commercial opportunities for advanced reproductive technologies in horses, wildlife, and companion animals. As advanced reproductive technologies become more efficient and repeatable in livestock and laboratory species, new opportunities will evolve to apply these techniques to alternative and non-traditional species. This will result in new markets requiring unique business models that address issues of animal welfare and consumer acceptance on a much different level than the livestock sector. Advanced reproductive technologies and genetic engineering will be applied to each species in innovative ways to provide breeders more alternatives for the preservation and propagation of elite animals in eac...
Characterization of the beta2-microglobulin gene of the horse. A clone containing beta(2)-microglobulin (beta(2)-m), the light chain of the major histocompatibility complex class I cell surface molecule, was isolated from an equine bacterial artificial chromosome library. This clone was used as a template for polymerase chain reaction (PCR) and unidirectional sequencing to elucidate the genomic sequence and intron/exon boundaries. We obtained 7,000 bases of sequence, extending from 1,100 nucleotides (nt) upstream of the coding region start through 1,698 nt downstream of the stop codon. The sequence contained regulatory elements in the region upstream of t...
Leptin in horses: tissue localization and relationship between peripheral concentrations of leptin and body condition. Obesity has been a major concern in the horse industry for many years, and the recent discovery of leptin and leptin receptors in numerous nonequine species has provided a basis for new approaches to study this problem in equine. The objectives were to: 1) clone a partial sequence ofthe equine leptin and leptin receptor genes so as to enable the design of primers for RT-PCR determination of leptin and leptin receptor gene presence and distribution in tissues, 2) develop a radioimmunoassay to quantify peripheral concentrations of leptin in equine, 3) determine if peripheral concentrations of le...
Molecular characterization of the equine testis-specific protein 1 (TPX1) and acidic epididymal glycoprotein 2 (AEG2) genes encoding members of the cysteine-rich secretory protein (CRISP) family. The cysteine-rich secretory protein (CRISP) family consists of three members called acidic epididymal glycoprotein 1 (AEG1), AEG2, and testis-specific protein 1 (TPX1), which share 16 conserved cysteine residues at their C-termini. The CRISP proteins are primarily expressed in different sections of the male genital tract and are thought to mediate cell-cell interactions of male germ cells with other cells during sperm maturation or during fertilization. Therefore, their genes are of interest as candidate genes for inherited male fertility dysfunctions and as putative quantitative trait loci fo...
Evaluation of a combined laser Doppler flowmetry and iontophoresis technique for the assessment of equine cutaneous microvascular function. A combined laser Doppler flowmetry and iontophoresis (LDFI) technique, used routinely to assess human microvascular function, was evaluated as a noninvasive technique for assessment of equine microvascular function, to facilitate the study of diseases such as laminitis. Baseline and vasoactive agonist-induced (acetylcholine and nitroprusside) microvascular flux was quantified at 2 sites (on the dorsal pastern adjacent to the coronary band and over the gluteals) in 6 clinically normal horses on 5 or 6 separate occasions under standardised conditions. Both agonists significantly increased microv...
Development and validation of a specific radioimmunoassay for equine osteocalcin. This study describes for the first time the development and validation of a sensitive and specific radioimmunoassay (RIA) for equine osteocalcin (OC) quantification using purified equine OC as standard, tracer, and immunogen for antibody formation in rabbits. The assay allowed to measure equine serum OC levels with a sensitivity of 0.2 ng/mL. Immunoreactive serum OC values of clinically normal, different-aged horses ranged from 3.68 to 127.31 ng/mL. Intra- and inter-assay coefficients of variation (CV) were 6.2 and 8.2%, respectively. Serial equine serum sample dilutions were linear. The recov...
Appearance of nitrite reducing activity of cytochrome c upon heat denaturation. The appearance of NO2- reducing activity of cytochrome c (Cyt c) upon heat denaturation was investigated with equine heart Cyt c. Denatured equine heart Cyt c (dCyt c), which was treated at 100 degrees C for 30 min, had NO2- reducing activity in the presence of dithionite and methylviologen in an aqueous solution under anaerobic conditions. In contrast, hemoglobin and myoglobin had no such activity under the same conditions. Using spectroscopic methods, we found that the appearance of this activity in the Cyt c was due to the following intramolecular changes: unfolding of the peptide chain, ex...
Expression of biologically active recombinant equine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae. The full-length equine interferon-gamma (eIFN-gamma) cDNA, including the secretion signal peptide coding region, was recloned into baculovirus transfer vector pAcYM1. This vector was co-transfected with Autographa californica nuclear polyhedrosis virus DNA or hybrid nuclear polyhedrosis virus DNA into Spodoptera frugiperda cells. The recombinant viruses, named AcEIFN-gamma and HyEIFN-gamma, were then recovered. Recombinant eIFN-gamma (reIFN-gamma) was accumulated in the culture fluid of the AcEIFN-gamma or HyEIFN-gamma infected Tricoplusia ni -derived cell line, BTI TN 5B1-4, and hemolymph of ...
Genetic characterization of horse bone excavated from the Kwakji archaeological site, Jeju, Korea. We determined the nucleotide sequences of the hypervariable D-loop region of mitochondrial DNA (mtDNA) from horse bone (humerus, A.D. 700 to A.D. 800) that was excavated from the Kwakji archaeological site, Jeju, Korea. We compared them with ones from extant horses. We designed three pairs of oligonucleotide primers from the tRNA-Thr and tRNA-Phe gene regions of mtDNA that are highly conserved among many other animal species. We cloned 232, 336, and 644 bp from the horse bone in order to determine the mtDNA D-loop sequence. The sequence was 1,124 bp long; the middle contained 19 tandem repeats...
Isolation of embryonic stem-like cells from equine blastocysts and their differentiation in vitro. Embryonic stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES-like cells from equine blastocysts that have been frozen and thawed. Our two lines of ES-like cells (E-1 and E-2) appear to maintain a normal diploid karyotype indefinitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage-specific embryonic antigen-1, STAT-3 and Oct 4. After culture of equine ES-like cells in vitro for more than 17 passages, some ES-like cells diffe...
Stabilization of protein by replacement of a fluctuating loop: structural analysis of a chimera of bovine alpha-lactalbumin and equine lysozyme. Equine lysozyme is a calcium-binding lysozyme and an evolutional intermediate between non-calcium binding c-type lysozyme and alpha-lactalbumin. We constructed a chimeric protein by substituting the fluctuating loop of bovine alpha-lactalbumin with the D-helix of equine lysozyme. The substitution affects the protection factors not only in the fluctuating loop but also in the antiparallel beta-sheet, the A- and B-helices, and the loop between the B-helix and the beta-sheet. Amide protons in these regions of the chimera are more protected from exchange than are those of bovine alpha-lactalbumin....
Induction of hyaluronan synthase 2 by human chorionic gonadotropin in mural granulosa cells of equine preovulatory follicles. In contrast to other species, the preovulatory rise in gonadotropins in mares causes a remarkable expansion of the entire granulosa cell layer in vivo, suggesting that hyaluronan (HA) synthesis may be regulated in mural granulosa cells in this species. The objectives of this study were to clone and characterize equine hyaluronan synthase 2 (HAS2) and investigate the regulation of its transcript and of HA synthesis in equine follicles during human chorionic gonadotropin (hCG)- induced ovulation. Results showed that the equine HAS2 cDNA contains a 5'-untranslated region of 436 bp, an open readin...
Construction of a stable non-mucoid deletion mutant of the Streptococcus equi Pinnacle vaccine strain. Streptococcus equi causes equine strangles, a purulent lymphadenopathy of the head and neck. An avirulent, non-encapsulated strain (Pinnacle) has been used widely in North America as an intranasal vaccine. The aim of the study was to create a specific mutation of the hyaluronate synthase (hasA) gene in Pinnacle to permanently abolish the production of capsule and provide an easily recognisable genetic marker. An internal fragment of hasA was generated by PCR and cloned into pTW100 (Microscience, UK). An encapsulated revertant of Pinnacle was then transformed with the recombinant plasmid by ele...
Surface plasmon resonance measurement of pH-induced responses of immobilized biomolecules: conformational change or electrostatic interaction effects? Recently, the observation of pH-induced conformational changes of biomolecules supported on carboxymethyldextran (CMD)-coated surfaces measured using surface plasmon resonance (SPR) has been reported. However, it is apparent that the evidence reported in the literature is ambiguous. The research presented in this paper describes investigations to study the changing SPR signal of immobilized biomolecules as a function of varying pH, to provide a detailed understanding of the origin of the pH-induced changes in the SPR profile. SPR measurements were performed with cytochrome c, concanavalin A, a...
Cloning and molecular characterization of an immunogenic LigA protein of Leptospira interrogans. A clone expressing a novel immunoreactive leptospiral immunoglobulin-like protein A of 130 kDa (LigA) from Leptospira interrogans serovar pomona type kennewicki was isolated by screening a genomic DNA library with serum from a mare that had recently aborted due to leptospiral infection. LigA is encoded by an open reading frame of 3,675 bp, and the deduced amino acid sequence consists of a series of 90-amino-acid tandem repeats. A search of the NCBI database found that homology of the LigA repeat region was limited to an immunoglobulin-like domain of the bacterial intimin binding protein of Esc...
Kinetic barriers to the folding of horse cytochrome C in the reduced state. To determine the kinetic barrier in the folding of horse cytochrome c, a CO-liganded derivative of cytochrome c, called carbonmonoxycytochrome c, has been prepared by exploiting the thermodynamic reversibility of ferrocytochrome c unfolding induced by guanidinium hydrochloride (GdnHCl), pH 7. The CO binding properties of unfolded ferrocytochrome c, studied by 13C NMR and optical spectroscopy, are remarkably similar to those of native myoglobin and isolated chains of human hemoglobin. Equilibrium unfolding transitions of ferrocytochrome c in the presence and the absence of CO observed by both e...
Cytogenetic localization of 136 genes in the horse: comparative mapping with the human genome. The aim of this study was to increase the number of type I markers on the horse cytogenetic map and to improve comparison with maps of other species, thus facilitating positional candidate cloning studies. BAC clones from two different sources were FISH mapped: homologous horse BAC clones selected from our newly extended BAC library using consensus primer sequences and heterologous goat BAC clones. We report the localization of 136 genes on the horse cytogenetic map, almost doubling the number of cytogenetically mapped genes with 48 localizations from horse BAC clones and 88 from goat BAC clon...
Characterization of the NRAMP1 (SLC11A1) gene in the horse (Equus caballus L.). The complete coding cDNA sequence of the horse NRAMP1 (SLC11A1) gene was determined (GenBank accession number AF354445). The nucleotide sequence of the horse NRAMP1 gene is similar to sequences of this gene in other species. The gene contains 15 exons whose total length of 1,635 bp corresponds to 544 amino acids constituting the resulting putative protein. Hydrophobicity profile analysis of the deduced horse NRAMP1 gene product showed a nearly identical structure with the mouse NRAMP1 protein. The gene was found to be located on the short arm of ECA 6p12-13 by fluorescence in situ hybridizatio...
Nucleation rate determination by a concentration pulse technique: application on ferritin crystals to show the effect of surface treatment of a substrate. The nucleation of horse spleen ferritin (HSF) crystals on substrates was investigated using a new modification of the double pulse technique. The influence of three different structureless substrates (glass, glass covered by methyl groups and poly-L-lysin template) on the nucleation was studied. The boundaries in the phase-diagram, which separate zones of crystal nucleation and growth were obtained by keeping pH = 5.0, and using CdSO(4) as crystallizing agent. The steady-state nucleation rates were determined. The energy required for critical nuclei formation was evaluated (10(-13) erg) and th...
Correlation between the osmotic second virial coefficient and solubility for equine serum albumin and ovalbumin. The Haas - Drenth - Wilson (HDW) (Haas et al., 1999) theoretical model was used to correlate osmotic second virial coefficient (B) values with solubility (S) values for equine serum albumin (ESA) and ovalbumin for corresponding solution conditions. The best fit from the theoretical model was compared to experimental S versus B data. B values were experimentally measured using static light scattering. Solubilities of ESA were estimated using a sitting drop method. When the experimental data for S versus B were plotted, an excellent fit for ESA was obtained according to the HDW model. The result...
Characterisation of the biological activity of recombinant equine eotaxin in vitro. The chemokine eotaxin (CCL11) is a key player in the trafficking of eosinophils to normal tissues and in the tissue eosinophilia associated with human allergic disease. We have recently cloned equine eotaxin and here we report the production of rEq eotaxin, with and without a C-terminal fusion peptide, in a novel expression system utilising stably transfected insect cells. rEq eotaxin induced equine eosinophil migration and superoxide production in vitro. A shape change in human eosinophils that could be blocked by 7B11, a monoclonal antibody against human CCR3, was also observed. Biological a...
Spectroscopic and electrochemical studies of horse myoglobin in dimethyl sulfoxide. This paper reports the first report of rapid, reversible direct electron transfer between a redox protein, specifically, horse myoglobin, and a solid electrode substrate in nonaqueous media and the spectroscopic (UV-vis, fluorescence, and resonance Raman) characterization of the relevant redox forms of myoglobin (Mb) in dimethyl sulfoxide (DMSO). In DMSO, the heme active site of metmyoglobin (metMb) appears to remain six-coordinate high-spin, binding water weakly. Changes in the UV-fluorescence spectra for metMb in DMSO indicate that the protein secondary structure has been perturbed and sugge...
