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.
Cloning of equine type II procollagen and the modulation of its expression in cultured equine articular chondrocytes. The complete nucleotide sequence of equine type II procollagen has not been previously reported, and equine-specific probes have not been available. We report the complete sequence and discuss the molecular characteristics of equine type II procollagen mRNA which was cloned from a cDNA library prepared from mRNA isolated from equine articular chondrocytes. The coding sequence (4257 bp) was 92.4% homologous to the cDNA of the human sequence, and the propeptide was 97% identical to the human sequence. We demonstrated that when equine chondrocytes are grown in phenotypically-maintained cultures, ...
Molecular cloning and functional expression of equine interleukin-1 receptor antagonist. Equine interleukin-1 receptor antagonist (IL-1ra) was molecularly cloned to establish a basis for cytokine therapy of acute and chronic inflammatory diseases in the horse. cDNA clones encoding the whole coding sequence of equine IL-1ra were isolated from equine peripheral blood mononuclear cells (PBMC) that had been stimulated with lipopolysaccharide (LPS). The equine IL-1ra cDNA obtained in this study contained an open reading frame encoding 177 amino acid residues. The predicted amino acid sequence of equine IL-1ra shared 75.7, 75.3 and 76.3% similarity with sequences of human, murine and ra...
Distribution and functional effects of neuropeptide Y on equine ureteral smooth muscle and resistance arteries. The distribution of neuropeptide Y (NPY)-immunoreactive (IR) nerves, as well as the functional effects of NPY and the Y1- and Y2-receptor agonists, [Leu31,Pro34]NPY and NPY(13-36), respectively, have been investigated in vitro in both visceral and arterial smooth muscle of the horse intravesical ureter. NPY-IR nerve fibres were widely distributed along the entire length of the ureter, although the intravesical part was the most richly innervated region, and the only one where NPY-IR ganglion cells were found. NPY (10(-7) M) did not affect either basal tone or spontaneous rhythmic contractions ...
Cloning, expression, purification, and characterization of the major core protein (p26) from equine infectious anemia virus. The gene coding for the major core protein (p26) of the lentivirus equine infectious anemia virus (EIAV) was cloned from EIAV infected serum, expressed in E. coli, and the resultant protein purified to electrophoretic homogeneity. The protein was expressed in a soluble form and was purified by conventional protein separation methods. When analyzed by SDS-PAGE, under both reducing and non-reducing conditions, the purified protein migrated as a 26 kDa monomer. Recombinant p26 (rp26), therefore, does not contain any intermolecular disulfide bond. Gel filtration chromatography also indicated that ...
A kinase-negative mutation of DNA-PK(CS) in equine SCID results in defective coding and signal joint formation. The equine SCID defect is more severe than its murine counterpart in that SCID foals are incapable of forming either coding or signal joints, whereas SCID mice manifest normal signal joint formation. To determine the basis of this difference and whether DNA-dependent kinase, catalytic subunit (DNA-PK(CS)), is involved in signal joint formation, equine DNA-PK(CS) transcripts were cloned and sequenced from normal and SCID cell lines. In the mutant allele, a frame-shift mutation truncates the protein N terminal of the domain with homology to the phosphatidylinositol 3-kinase family resulting in c...
Molten globule state of equine beta-lactoglobulin. The acid-unfolded state of equine beta-lactoglobulin was characterized by means of circular dichroism, nuclear magnetic resonance, analytical gel-filtration chromatography, and analytical centrifugation. The acid-unfolded state of equine beta-lactoglobulin has a substantial secondary structure as shown by the far-ultraviolet circular dichroism spectrum but lacks persistent tertiary packing of the side chains as indicated by the near-ultraviolet circular dichroism and nuclear magnetic resonance spectra. It is nearly as compact as the native conformation as shown by the gel filtration and sedime...
Partial cloning of prohibitin cDNA from canine, feline, bovine, equine, and rabbit liver mRNA by RT-PCR. Prohibitin is the protein which has an inhibitory function in cell growth, and its gene is suggested to be one of putative tumor suppressor genes. In this report, we described a partial cloning of prohibitin cDNAs from canine, feline, bovine, equine, and rabbit liver mRNAs by RT-PCR, and their homology analysis. The sequences of these RT-PCR products were compared with each other as well as those reported for human and rat. The homology in this region of prohibitin cDNA was approximately 90%, and the amino acid sequence of each RT-PCR product shared more than 95% identity. Therefore, it is con...
An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription. Equine arteritis virus (EAV) is a positive-strand RNA virus that uses a discontinuous transcription mechanism to generate a nested set of six subgenomic mRNAs from which its structural genes are expressed. A stable bacterial plasmid (pEAV030) containing a full-length cDNA copy of the 12.7-kb EAV genome was constructed. After removal of a single point mutation in the replicase gene, RNA transcripts generated in vitro from pEAV030 were shown to be infectious upon electroporation into BHK-21 cells. A genetic marker mutation was introduced at the cDNA level and recovered from the genome of the pro...
Quantitative ionspray liquid chromatographic/tandem mass spectrometric determination of reserpine in equine plasma. A method based on ionspray liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed for the determination of reserpine in equine plasma. A comparison was made of the isolation of reserpine from plasma by liquid-liquid extraction and by solid-phase extraction. A structural analog, rescinnamine, was used as the internal standard. The reconstituted extracts were analyzed by ionspray LC/MS/MS in the selected reaction monitoring (SRM) mode. The calibration graph for reserpine extracted from equine plasma obtained using liquid-liquid extraction was linear from 10 to 5000 pg ml-1 and t...
Dose-response of X-irradiated human and equine lymphocytes. We have investigated and compared DNA damage and cell killing induced in human and equine lymphocytes after in vitro X-irradiation. Our data show that the cytogenetic and the lethality effects are both greater in equine lymphocytes, but that the difference is wider for lethality. The ratios between doses inducing the same effect are 1.3, 1.7 and 9.4 for the number of binucleated cells with micronuclei, micronucleus frequency in binucleated cells and DNA synthesis inhibition, respectively. The very different radiosensitivity observed for the two mammalian species encourages us to use their lymp...
Altered biological activity of equine chondrocytes cultured in a three-dimensional fibrin matrix and supplemented with transforming growth factor beta-1. To determine the effects of transforming growth factor-beta 1 (TGF-beta 1) on the synthesis of DNA, collagen, and proteoglycans (PG) by equine chondrocytes. Methods: Articular cartilage obtained from multiple joints of a 4-month-old foal. Methods: Chondrocytes were isolated by collagenase digestion, cultured in monolayer, trypsinized, and implanted at a cellular density of 10 x 10(6) chondrocytes/ml in a three-dimensional fibrin matrix. Chondrocytes in culture were supplemented with TGF-beta 1 at concentrations of 0, 1, 5, or 10 ng/ml in serum-free medium or medium containing fetal bovine seru...
Heart rate variability in the horse by ambulatory monitoring. Using a microprocessor controlled Ambulatory Monitoring System (AMS) developed by one of us (LvD), we have been studying the changes in and control of heart rate in the resting horse. The system provides us with InterBeat Intervals (IBI in milliseconds), motion sensing, and a time domain measure (mean successive differences: MSD) of heart rate variability for periods up to 72 hours. Thoracic impedance is also available but parameters for the equine chest are not currently available. The system is completely noninvasive, small, and carried on a surcingle worn by the subject. The equine subject ...
Myoglobin oxygen dissociation by multiwavelength spectroscopy. Multiwavelength optical spectroscopy was used to determine the oxygen-binding characteristics for equine myoglobin. Oxygen-binding relationships as a function of oxygen tension were determined for temperatures of 10, 25, 35, 37, and 40 degrees C, at pH 7.0. In addition, dissociation curves were determined at 37 degrees C for pH 6.5, 7.0, and 7.5. Equilibration was achieved with a myoglobin solution, at the desired temperature and pH, and 16 oxygen-nitrogen gas mixtures of known oxygen fraction. Correction for the inevitable presence of metmyoglobin was made by using a three-component least squ...
Characterization of horse (Equus caballus) immunoglobulin mu chain-encoding genes. Horse (Equus caballus) immunoglobulin mu chain-encoding (IgM) variable, joining, and constant gene segments were cloned and characterized. Nucleotide sequence analyses of 15 cDNA clones from a mesenteric lymph node library identified 7 unique variable gene segments, 5 separate joining segments, and a single constant region. Based on comparison with human sequences, horse variable segments could be grouped into either family 1 of immunoglobulin (Ig) clan I or family 4 of Ig clan II subclan IV. All horse sequences had a relatively conserved 16 base pair (bp) segment in framework 3 which was reco...
Identification, cloning and sequence analysis of the equine adenovirus 1 hexon gene. Based on sequence homology with human adenovirus 2 (HAdV2), the hexon gene of equine adenovirus 1 (EAdV1) was identified. HindIII restriction fragments containing the hexon and other viral genes were cloned into the plasmids pUC19 and pBlueScript SK(-) and sequenced. The nucleotide sequence of the hexon gene was completely determined and partial sequence data were obtained for seven other EAdV1 genes. Amino acid (aa) sequence comparison with published adenovirus (AdV) proteins identified the genes for the IIIa, penton, pVII, PVI, 23K proteinase, DNA binding and 100K proteins. The eight EAdV1 g...
Local electrostatic potentials in pyridoxal phosphate labelled horse heart cytochrome c. The present work shows the application of an optical label pyridoxal phosphate (PLP) for the experimental determination of local electrostatic potentials in singly substituted cytochromes c modified by pyridoxal phosphate at Lys 79 (PLP-Lys-79-cyt.c) or at Lys 86 (PLP-Lys-86-cyt.c). PLP has also been used to calculate the pKa values of all ionizable groups and the electrostatic potentials in the modified proteins and to analyse their properties. The experimental pKa values for the pyridine nitrogen and phenolic hydroxyl of the bound label were obtained from pH-dependent absorbance and fluoresc...
Expression of equine morbillivirus (EMV) matrix and fusion proteins and their evaluation as diagnostic reagents. Full-length cDNA clones coding for the matrix (M) and fusion (F) proteins of equine morbillivirus (EMV) were isolated by RT-PCR, and expressed in Escherichia coli using two different expression systems. Western blot analysis indicated that the M and F proteins, expressed either by itself or as fusion proteins with glutathione S-transferase (GST), were insoluble and degraded after expression. Analysis of the degradation pattern of recombinant M protein suggested that the N-terminus of the matrix protein might be more stable and antigenic than the C-terminal region. Therefore a third system was ...
cDNA cloning and sequencing reveal the major horse allergen Equ c1 to be a glycoprotein member of the lipocalin superfamily. The gene encoding the major horse allergen, designated Equus caballus allergen 1 (Equ c1), was cloned from total cDNA of sublingual salivary glands by reverse transcription-polymerase chain reaction using synthetic degenerate oligonucleotides deduced from N-terminal and internal peptide sequences of the glycosylated hair dandruff protein. A recombinant form of the protein, with a polyhistidine tail, was expressed in Escherichia coli and purified by immobilized metal affinity chromatography. The recombinant protein is able to induce a passive cutaneous anaphylaxis reaction in rat, and it behave...
Collagen fiber organization is related to mechanical properties and remodeling in equine bone. A comparison of two methods. We studied birefringence as an indicator of collagen fiber orientation in the diaphysis of the equine third metacarpal bone. We had previously shown that tissue from the lateral cortex of this bone is stronger monotonically, but less fatigue resistant, than tissue from the medial and dorsal regions. To learn whether collagen fiber orientation might play a role in this regional specialization, we tested three hypotheses using the same specimens: (1) collagen fiber orientation is regionally dependent; (2) remodeling changes collagen fiber orientation; (3) longitudinal collagen fibers correlate p...
Purification, crystallization and preliminary crystallographic analysis of mare lactoferrin. Lactoferrin is an iron-binding glycoprotein with a molecular weight of 80 kDa. The protein has two iron binding sites. It has two structural lobes, each housing one Fe(3+) and the synergistic CO(3)(2-) ion. The protein was isolated from the colostrum/milk of mares maintained at National Research Centre on Equines, Hisar, India. The purified samples of the protein were crystallized using a microdialysis method. The protein was dialysed against low ionic strength buffer solution. Several crystal forms were obtained, out of which three were characterized which have cell dimensions as follows. For...
Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin expression for enhancing Escherichia coli growth in a microaerobic bioreactor. Expression of a gene encoding hemoglobin (VHb) from the aerobic bacterium Vitreoscilla sp. in several organisms, including Escherichia coli, has been shown to improve microaerobic cell growth and enhance oxygen-dependent product formation. The suitability of VHb to enhance microaerobic metabolism has been suggested to depend on its unusual oxygen binding characteristics. To examine whether hemoproteins of other origins can also elicit the positive effects VHb exerts in microaerobic E. coli cells, we subcloned the genes encoding Vitreoscilla VHb, horse heart myoglobin (HMb), and yeast flavohemo...
Equine motor neuron disease is not linked to Cu/Zn superoxide dismutase mutations: sequence analysis of the equine Cu/Zn superoxide dismutase cDNA. The cDNA encoding the equine copper/zinc superoxide dismutase (SOD1) was cloned from leukocyte total RNA from healthy horses and its nucleotide (nt) sequence was determined. We further sequenced the SOD1 gene from 16 horses diagnosed with equine motor neuron disease (EMND) and eight unrelated, clinically normal horses to determine if this disease, similar to amyotrophic lateral sclerosis (ALS) in humans, is linked to SOD1 mutations. The 465-bp SOD1 coding region in the horse encodes 153 amino acid (aa) residues. Equine SOD1 exhibited 81.8 and 79.9% sequence identity to the human homolog at the...
Interaction of GroEL with conformational states of horse cytochrome c. GroEL interacts with proteins in denatured states and promotes their efficient folding. To understand the conformational features required for the substrate, we studied the interactions of GroEL with various derivatives of horse cytochrome c including porphyrin-cytochrome c, apo-cytochrome c, and the three fragments containing the heme group, i.e. fragments 1-65, 1-38 and 11-21. Size-exclusion chromatography was performed, taking advantage of the heme absorption of the fluorescence label. Under low-salt conditions, significant binding to GroEL was observed for porphyrin-cytochrome c, apo-cytoc...
Variability of cardiomyocyte DNA content, ploidy level and nuclear number in mammalian hearts. DNA content, ploidy level, cell size and nuclear number were investigated in 54 mammalian hearts from nine species. DNA content was determined biochemically and ploidy level of cells was studied by the means of Feulgen cytophotometry. Nuclear number was calculated by a new method, while cell size was determined by using ocular micrometry. In most mammals diploid cell nuclei predominate. Higher ploidy levels were found in the human and the pig hearts. The total amount of DNA correlated with the myocardial weight. Eight million heart muscle cell nuclei were found in mice (myocardial weight 160 m...
Magnetic motor evoked potentials in ponies. Magnetic stimulation of motor pathways was used to effect motor unit action potential recordings from forelimb and hindlimb muscles in unanesthetized ponies. Motor pathway conduction velocities to the forelimb and hindlimb were determined to be 53.8 +/- 9.6 m/s-1 and 63.4 +/- 8.3 m/s-1, respectively. This noninvasive technique will enable more precise evaluation of motor deficits in clinical patients than is possible with the neurological examination.
Molecular diffusion into horse spleen ferritin: a nitroxide radical spin probe study. Electron paramagnetic resonance spectroscopy and gel permeation chromatography were employed to study the molecular diffusion of a number of small nitroxide spin probes (approximately 7-9 A diameter) into the central cavity of the iron-storage protein ferritin. Charge and polarity of these radicals play a critical role in the diffusion process. The negatively charged radical 4-carboxy-2,2,6,6-tetramethylpiperidine-N-oxyl (4-carboxy-TEMPO) does not penetrate the cavity whereas the positively charged 4-amino-TEMPO and 3-(aminomethyl)-proxyl radical and polar 4-hydroxy-TEMPO radical do. Unlike th...
Tumour suppressor gene p53 in the horse: identification, cloning, sequencing and a possible role in the pathogenesis of equine sarcoid. The tumour suppressor protein p53 enhances the genetic stability of the cell and plays a critical role in tumour suppression. Equine p53 was analysed by sequencing exons 5 to 9, a region which includes most known mutations and all the mutational hotspots in the species that have been investigated. The fragment was amplified, cloned and sequenced from genomic and complementary DNA. A comparison of the predicted amino acid sequences between the horse and other species resulted in identities between 66 per cent with the clawed frog and 92 per cent with the cat. Using the single strand conformatio...
Assessment of autonomic nervous function by power spectral analysis of heart rate variability in the horse. We studied power spectral analysis of heart rate (HR) variability in the horse, with the hypothesis that the quantitative information provided by the spectral analysis of HR variability reflects the interaction between sympathetic and parasympathetic regulatory activities. For this purpose, electrocardiogram, blood pressure (BP) and respiratory (Resp) waveform were simultaneously recorded from Thoroughbred horses (3-5 years old) and analyzed by power spectrum. There were two major spectral components at low-frequency (LF) and high-frequency (HF) bands for HR variability. The peak of Resp varia...