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Topic:Biochemistry
The study of biochemistry in horses encompasses the chemical processes and substances that occur within equine organisms. This field investigates the molecular interactions and pathways that are fundamental to horse physiology, including metabolism, enzyme activity, and genetic expression. Key areas of interest include the examination of metabolic disorders, nutrient absorption, and the biochemical basis of muscle function and energy production. Researchers utilize biochemical analysis to understand health and disease mechanisms in horses, contributing to the development of diagnostic tools and therapeutic strategies. This page gathers peer-reviewed studies and scholarly articles that explore various biochemical processes and their implications for equine health and performance.
Expression of biologically active recombinant equine interferon-gamma in Escherichia coli. Interferon gamma (IFN-gamma) is a pleiotropic cytokine that is recognized as an important modulator of the immune response. To date, there is no report that prokaryocyte-derived recombinant equine IFN-gamma has antiviral activity. In this report, the gene coding equine IFN-gamma (EIFN-gamma) mature protein was cloned into pET-28a (+) and the recombinant EIFN-gamma was expressed in Escherichia coli (E. coli). The antiviral activity of expressed recombinant EIFN-gamma was evaluated by using a recombinant Vesicular Stomatitis Virus expressing green fluorescence protein (rVSV-GFP) system in the eq...
Activated platelet-derived growth factor beta receptor expression, PI3K-AKT pathway molecular analysis, and transforming signals in equine sarcoids. The equine sarcoid is the most common dermatologic neoplasm reported in horses. Bovine papillomavirus (BPV) types 1 and 2 are associated with sarcoids, in which the expression of the major transforming oncoprotein (E5) is often recorded. The transformation activity of the virus is due to the binding of the E5 to the platelet-derived growth factor beta receptor (PDGFbeta-r). In the present study, we show by Western blot in 4 sarcoid samples and 3 normal equine skin samples that the PDGFbeta-r is more phosphorylated in sarcoid tissue than in normal skin (P < .001). Furthermore, the physical i...
Feeding grape seed extract to horses: effects on health, intake and digestion. A feeding trial involving four Thoroughbred race horses was undertaken to establish whether inclusion of grape seed extract (GSE) in the diet of horses undergoing mild exercise had any effects on their general health, intake and digestion. Supplementation with GSE had no effect on either feed or water intake of the horses and the supplement was readily palatable to the horses at all levels of inclusion. Feeding GSE caused no adverse effects in terms of animal health (temperature, pulse and respirations rates), and there were some positive effects related to a presumed alteration in fermentatio...
Evaluation of topically administered diclofenac liposomal cream for treatment of horses with experimentally induced osteoarthritis. To assess the clinical, biochemical, and histologic effects of topically administered diclofenac liposomal cream (DLC) in the treatment of horses with experimentally induced osteoarthritis. Methods: 24 horses. Methods: Osteoarthritis was induced arthroscopically in 1 middle carpal joint of all horses. Eight horses treated with DLC were given 7.3 g twice daily via topical application. Eight horses treated with phenylbutazone were given 2 g orally once daily. Eight control horses received no treatment. Evaluations included clinical, radiographic, magnetic resonance imaging, synovial fluid, gross...
Equine cytochrome P450 2C92: cDNA cloning, expression and initial characterization. Substantial gaps exist in our knowledge of the metabolic clearance of therapeutic agents in horses. Accordingly, a cytochrome P450 monooxygenase in the 2C family was cloned from an equine liver, sequenced and expressed in a baculovirus expression system. Catalytic activities of the recombinant protein were measured with a number of substrates. The protein, assigned CYP2C92, displayed optimal catalytic activity with diclofenac using molar ratios of CYP2C92 to NADPH CYP450 reductase of 1:18. Addition of cytochrome b(5) to diclofenac incubations had no significant effect on metabolic turnover. CY...
Evaluation of polysulfated glycosaminoglycan or sodium hyaluronan administered intra-articularly for treatment of horses with experimentally induced osteoarthritis. To assess clinical, biochemical, and histologic effects of polysulfated glycosaminoglycan (PSGAG) or sodium hyaluronan administered intra-articularly in treatment of horses with experimentally induced osteoarthritis. Methods: 24 horses. Methods: Osteoarthritis was induced arthroscopically in 1 middle carpal joint of all horses. Eight horses received hyaluronan (20 mg) and amikacin (125 mg) intra-articularly on study days 14, 21, and 28. Eight horses received PSGAG (250 mg) and amikacin (125 mg) intra-articularly on study days 14, 21, and 28. Eight control horses received 2 mL of saline (0.9% N...
A multifrequency HYSCORE study of weakly coupled nuclei in frozen solutions of high-spin aquometmyoglobin. In this work, we show the extreme power of multifrequency HYSCORE (hyperfine sublevel correlation spectroscopy) techniques to unravel the hyperfine interactions of the electron spin with the remote nuclei in the heme site of high-spin ferric heme proteins. Horse heart aquo-metmyoglobin was used as a model system to demonstrate the power of these techniques. Experimental evidence was collected and assigned to protons of the proximal histidine ligand, to the mesoprotons of the heme ligand, and to two different protons of the distal water ligand. The latter difference relates to the stabilization...
Development of an intra-lamellar microdialysis method for laminitis investigations in horses. Microdialysis (MD) was used for continuous monitoring of the lamellar extracellular fluid (ECF) in six mature healthy Standardbred horses. MD probes were introduced into the lamellar tissue under local anaesthesia. Following intravenous injection of gentamicin (5mg/kg), MD and serum samples were collected for 24h and analysed using a sensitive ELISA test for gentamicin and fluorescence polarization immunoassay for urea concentrations. Calibration of probes was performed through in vivo urea recovery and in vitro gentamicin and urea recovery. Data obtained from different body compartments were ...
Effects of compounding and storage conditions on stability of pergolide mesylate. To determine the effects of temperature and light over a 35-day period on stability of pergolide mesylate after compounding in an aqueous vehicle. Methods: Evaluation study. Methods: Pergolide was compounded into a formulation with a final target concentration of 1 mg/mL. Aliquots of the formulation were then stored at -20 degrees, 8 degrees, 25 degrees, or 37 degrees C without exposure to light or at 25 degrees C with exposure to light for 35 days. Samples were assayed in triplicate by means of high-pressure liquid chromatography immediately after compounding and after 1, 7, 14, 21, and 35 da...
In vivo and in vitro effects of interleukin-1beta on equine oocyte maturation and on steroidogenesis and prostaglandin synthesis in granulosa and cumulus cells. We analysed the effect of interleukin-1 on oocyte maturation and on steroid and prostaglandin production by equine granulosa and cumulus cells. In Experiment 1, interleukin-1beta (IL-1beta) was injected into the growing dominant follicle, which was punctured 38 h later. Follicular fluid was assayed for steroids and prostaglandin-F2alpha (PGF2alpha). Granulosa cells were analysed for 3beta-hydroxysteroid dehydrogenase (3beta-HSD), progesterone receptor (PR), cyclooxygenase 1 and 2 (Cox 1 and Cox 2) and steroidogenic acute regulatory protein (StAR) mRNAs. In Experiment 2, cumulus-oocyte complexe...
Identification of protein tyrosine phosphatases and dual-specificity phosphatases in mammalian spermatozoa and their role in sperm motility and protein tyrosine phosphorylation. Protein tyrosine kinases have important roles in spermatozoa; however, little is known about the presence and regulation in these cells of their counterparts in signaling, namely, protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). The objectives of the present study were to identify PTPs and DSPs in boar, stallion, and dog spermatozoa; to characterize their subcellular distribution; and to investigate the roles of tyrosine phosphatases in maintenance of protein tyrosine phosphorylation level and in sperm motility. Using Western blotting with specific antibodies in b...
The Streptococcus equi prophage-encoded protein SEQ2045 is a hyaluronan-specific hyaluronate lyase that is produced during equine infection. Streptococcus equi causes equine 'strangles'. Hyaluronate lyases, which degrade connective tissue hyaluronan and chondroitins, are thought to facilitate streptococcal invasion of the host. However, prophage-encoded hyaluronate lyases are hyaluronan-specific and are thought to be primarily involved in the degradation of the hyaluronan capsule of streptococci during bacteriophage infection. To understand the role of prophage-encoded hyaluronate lyases further, we have biochemically characterized such a hyaluronate lyase, SEQ2045 from S. equi, and have shown that it is produced during equine infe...
Hyperactivation of stallion sperm is required for successful in vitro fertilization of equine oocytes. Capacitation is a complex and not well-understood process that encompasses all the molecular changes sperm must undergo to successfully fertilize an oocyte. In vitro fertilization has remained elusive in the horse, as evidenced by low in vitro fertilization (IVF) rates (0%-33%); moreover, only two foals have ever been produced using IVF. Incubation of stallion sperm in modified Whittens supplemented with bovine serum albumin and sodium bicarbonate yielded significant rates of time-dependent protein tyrosine phosphorylation and induced acrosomal exocytosis, consistent with capacitation. The obj...
Can programmed cell death be induced in post-ejaculatory bull and stallion spermatozoa? Apoptosis is common during spermatogenesis. Here, it was tested whether apoptosis could be induced in sperm after ejaculation. There were several lines of evidence to indicate that sperm are resistant to induction of apoptosis. First, incubation of bull sperm at temperatures characteristic of normothermia (38.5 degrees C) or heat shock (40 and 41 degrees C) for 4h did not increase the proportion of sperm positive for the TUNEL reaction. There was also no reduction in mitochondrial polarity caused by exposure to 40 or 41 degrees C. Incubation at 38.5 degrees C (least-squares mean+/-SEM=4.0+/-1....
Screening, quantification, and confirmation of phenylbutazone and oxyphenbutazone in equine plasma by liquid chromatography-tandem mass spectrometry. A sensitive liquid chromatographic-tandem mass spectrometric method was developed and validated for screening, quantification, and confirmation of phenylbutazone and oxyphenbutazone in equine plasma. Analytes were recovered from plasma by liquid-liquid extraction followed by separation in a reversed-phase column and identification by mass spectrometry with selected reaction monitoring in negative electrospray ionization mode. Extraction recovery for both analytes was >80%. Limits of detection, quantification, and confirmation for both analytes were 0.01 microg/mL (S/N>or= 3), 0.05 microg...
Use of a bacteriophage lysin, PlyC, as an enzyme disinfectant against Streptococcus equi. Streptococcus equi is the causative agent of the purulent infection equine strangles. This disease is transmitted through shedding of live bacteria from nasal secretions and abscess drainage or by contact with surfaces contaminated by the bacteria. Disinfectants are effective against S. equi, but inactivation by environmental factors, damage to equipment, and toxicity are of great concern. Bacteriophage-encoded lysins (cell wall hydrolases) have been investigated as therapeutic agents due to their ability to lyse susceptible gram-positive organisms. Here, we investigate the use of one lysin, P...
Response of biochemical markers of bone metabolism to exercise intensity in thoroughbred horses. We studied the response of biochemical markers of bone metabolism to exercise intensity in horses. Four horses were walked on a mechanical walker for one week (pre-exercise). Then they performed low-speed exercise on a high-speed treadmill in the first week and medium-speed exercise in the second week and high-speed exercise in the third week of training. We measured two indices of bone resorption, serum hydroxyproline concentration and the urinary deoxypyridinoline/creatinine ratio, and serum osteocalcin (OC) concentration as an index of bone formation. Both indices of bone resorption gradual...
Antibody immobilization on to polystyrene substrate–on-chip immunoassay for horse IgG based on fluorescence. A simple microfluidic immunoassay card was developed based on polystyrene (PS) substrate for the detection of horse IgG, an inexpensive model analyte using fluorescence microscope. The primary antibody was captured onto the PS based on covalent bonding via a self-assembled monolayer (SAM) of thiol to pattern the surface chemistry on a gold-coated PS. The immunosensor chip layers were fabricated from sheets by CO(2) laser ablation. The functionalized PS surfaces after each step were characterized by contact angle measurement, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (...
Human osteoclast formation and activity on an equine spongy bone substitute. The aim of the present study was to evaluate the in vitro formation and activity of human osteoclasts (OCLs) generated on a new type of xenograft for bone substitution, an equine spongy bone. Methods: Peripheral blood mononuclear cells from healthy volunteers were used to generate OCLs in vitro in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) on bovine bone slices (positive control) and equine spongy bone. Morphological and biochemical methods were used to assess OCLs formation and activity. Results: Cells generated after 21 day...
Interleukin-1 receptor antagonist expression in the equine endometrium during the peri-implantation period. To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was fou...
Modification of equine sperm chromatin decondensation method to use fluorescence in situ hybridization (FISH). Fluorescence in situ hybridization (FISH) is widely used in the study of chromosome structure and organization. Cytogenetic evaluation of chromosomes using FISH technique plays an increasingly important role in diagnosing karyotype changes in both somatic and reproductive cells. The aim of the study was to optimize the conditions of stallion sperm decondensation, which have a significant effect on the results of fluorescence in situ hybridization. Appropriate type and time of decondensation was chosen for the sperm of every stallion. It was found that decondensation performed using a preparati...
Rapid calcitonin response to experimental hypercalcemia in healthy horses. Calcium has important physiological functions, and disorders of calcium homeostasis are frequent in horses. We have made important progress understanding equine calcium homeostasis; however, limited information on equine calcitonin (CT) is available, in part because of the lack of validated CT assays. To determine the CT response to high ionized calcium (Ca(2+)) concentrations in healthy horses, we induced hypercalcemia in 10 healthy horses using a calcium gluconate 23% solution (5mg/kg; 120 mL/500 kg horse) infused over 4 min. Four horses were infused with 120 mL of 0.9% NaCl and used as cont...
Effects of competition experience and transportation on the adrenocortical and thyroid responses of horses. To evaluate whether the amount of experience of sport horses and the stress of transport affected their adrenocortical and thyroid responses, the plasma concentrations of total cortisol and total and free iodothyronine of 63 horses were studied before and after show jumping competitions. There were 14 trained inexperienced jumpers (group 1), 20 trained experienced jumpers (group 2), 10 trained inexperienced jumpers that had been transported just before the competition (group 3) and 19 trained experienced jumpers that had been transported just before the competition (group 4). The concentration...
Transient increase with strenuous exercise of plasma levels of glycosaminoglycans in humans and horses. Plasma glycosaminoglycans (GAGs) were isolated and purified by chromatographic procedures in healthy humans and horses before and after physical exercise. A weak anion exchange resin was used to separate polyanions. Humans exercised on a cycloergometer, while horses were exercised on a treadmill and in show jumping competition. Some GAGs were isolated from untreated plasma and operationally defined as native proteoglycans (PGs), while the total GAG amount was isolated from mild alkali treated plasma. Plasma GAG concentrations in terms of galactosamine and galactose, the respective components o...
Evaluation of tramadol and its main metabolites in horse plasma by high-performance liquid chromatography/fluorescence and liquid chromatography/electrospray ionization tandem mass spectrometry techniques. Tramadol is a centrally acting analgesic drug that has been used clinically for the last two decades to treat pain in humans. The clinical response of tramadol is strictly correlated to its metabolism, because of the different analgesic activity of its metabolites. O-Desmethyltramadol (M1), its major active metabolite, is 200 times more potent at the micro-receptor than the parent drug. In recent years tramadol has been widely introduced in veterinary medicine but its use has been questioned in some species. The aim of the present study was to develop a new sensible method to detect the whole ...
Antigenic, microbicidal and antiparasitic properties of an l-amino acid oxidase isolated from Bothrops jararaca snake venom. Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800mug/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal compon...
Assessment of the transformation of equine skin-derived fibroblasts to multinucleated skeletal myotubes following lentiviral-induced expression of equine myogenic differentiation 1. To develop a reliable method for converting cultured equine skin-derived fibroblasts into muscle cells. Methods: Equine skin-derived fibroblasts. Methods: The equine myogenic differentiation 1 (eqMyoD) genomic sequence was obtained by use of equine bacterial artificial chromosome screening and PCR sequencing. Total mRNA was extracted from foal skeletal muscle, and eqMyoD cDNA was cloned into a plasmid vector with an internal ribosomal entry site to express bicistronic eqMyoD or enhanced green fluorescent protein (EGFP). Transient expression was confirmed by immunocytochemical analysis and west...
Effects of intravenous administration of caffeine on physiologic variables in exercising horses. To investigate the effect of acute administration of caffeine on the athletic performance of Arabian horses. Methods: 12 healthy adult Arabian horses that were trained for exercise on a treadmill. Methods: By use of a crossover study design, horses received each of the following treatments: IV administration of caffeine (5 mg/kg) and IV administration of approximately the same volume of saline (0.9% NaCl) solution. Order of treatment was randomized, and there was a 10-day interval between treatments. Thirty minutes after treatments, horses underwent an incremental exercise test (IET) on a trea...
Matrix metalloproteinases in inflammatory pathologies of the horse. The extracellular matrix (ECM) of connective tissue is constantly being remodelled to allow for growth and regeneration. Normal tissue maintenance requires the ECM components to be degraded and re-synthesised in relatively equal proportions. This degradation is facilitated by matrix metalloproteinases (MMPs) and their proteolytic action is controlled primarily by the tissue inhibitors of metalloproteinases (TIMPs). Both MMPs and TIMPs exist in a state of dynamic equilibrium, with a slight excess of one or the other depending on the need for either ECM breakdown or synthesis. Long-term disrupti...
Comparative immunolocalization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa. Spermatozoa, as other eukaryotic cells, need hexoses to produce energy to maintain membrane homeostasis, to move along the female genital tract and to carry the male genome to the female gamete. GLUTs are a family of proteins that permit and improve the passive transport of hexoses inside cells. This study was aimed at investigating the presence and localization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa by both immunofluorescence and western blotting. GLUTs exhibited a peculiar distribution along the sperm cell depending on the isoforms considered, the hexose they transport ...
