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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.
A native disulfide stabilizes non-native helical structures in partially folded states of equine β-lactoglobulin. Equine β-lactoglobulin (ELG) assumes non-native helices during refolding and in partially folded states. Previously, circular dichroism (CD) combined with site-directed mutagenesis identified helical regions in the acid- and cold-denatured states of ELG. It is also known that a fragment of ELG, CHIBL (residues 88-142), has a structure similar to that of the cold-denatured state. For the study reported herein, the structure of a shorter fragment, CHIBLΔF (residues 97-142), was investigated by CD and nuclear magnetic resonance spectroscopy. The secondary chemical shifts clearly showed that non...
Consequences of butylated hydroxytoluene in the freezing extender on post-thaw characteristics of stallion spermatozoa in vitro. Ejaculates from six pure Spanish stallions were split, and one subsample frozen in a commercial extender supplemented with the lipid soluble antioxidant butylated hydroxytoluene (BHT), while the other subsample served as control. After at least 4 weeks of storage, samples were thawed and post-thaw sperm quality analysed: sperm motility and kinematics using a CASA system, membrane and acrosome integrity and mitochondrial membrane potential using flow cytometry. The outcome of cryopreservation varied significantly among stallions. However, the supplementation with 1 mm BHT had no significant eff...
Validation of a liquid chromatography-tandem mass spectrometry method for quantification of glycopyrrolate in horse plasma. A rapid, sensitive, and specific ultra-high-performance liquid chromatography with heated electrospray ionization-tandem mass spectrometry (UHPLC-HESI-MS-MS) method to detect and quantify glycopyrrolate in horse plasma has been developed and validated. We also determined glycopyrrolate in plasma after oral and intravenous administration of clinically relevant doses to Thoroughbred horses. Calibration was accomplished by weighted, linear regression analysis using a deuterated analogue of glycopyrrolate as internal standard (IS). Glycopyrrolate (GLY) and the IS (GLY-d(3)) were isolated from plas...
GTG mutation in the start codon of the androgen receptor gene in a family of horses with 64,XY disorder of sex development. Genetic sex in mammals is determined by the sex chromosomal composition of the zygote. The X and Y chromosomes are responsible for numerous factors that must work in close concert for the proper development of a healthy sexual phenotype. The role of androgens in case of XY chromosomal constitution is crucial for normal male sex differentiation. The intracellular androgenic action is mediated by the androgen receptor (AR), and its impaired function leads to a myriad of syndromes with severe clinical consequences, most notably androgen insensitivity syndrome and prostate cancer. In this paper, w...
A pilot study on ethanol-polyethylene glycol-formalin fixation of farm animal cadavers. Most embalming of cadavers for anatomical dissection in veterinary medicine has used 6-10% formaldehyde resulting in discoloured and rigid specimens. This project produced teaching specimens of sheep, horse and calf cadavers having their musculoskeletal and visceral structures with a natural appearance using a fixation solution with lowered concentrations of formaldehyde (2% and 3%) together with ethanol and polyethylene glycols. Fixation parameters (palpable consistency, flexibility, colour, tissue hydration and odour) were assessed qualitatively by twice weekly dissections over two months fo...
Characterization of L-lysine transport across equine and porcine jejunal and colonic brush border membrane. In nonruminant herbivores, microbially derived AA could contribute to whole-body AA homeostasis and thus decrease predicted AA requirements. However, postileal capacity of AA uptake is currently unknown. Therefore, to test the hypothesis that Lys is transported across the large colon mucosal apical membrane with capacity similar to that of the small intestinal mucosa in the pony and pig, we examined Lys transport in vitro using brush border membrane vesicles (BBMV). Mucosa was collected from the distal jejunum (DJ) and proximal large colon (PLC) of growing pigs (n = 3) and ponies (n = 4), flas...
Overexpression of histidine-rich calcium binding protein in equine ventricular myocardium. Histidine-rich calcium binding protein (HRC) is a high capacity, low affinity Ca(2+) binding protein, specifically expressed in striated muscles of mammals. In rabbit skeletal and cardiac muscles, HRC binds to sarcoplasmic reticulum (SR) membranes via triadin, a junctional SR protein. Recently, a potential role in heart failure and arrhythmogenesis has been assigned to HRC due to its activity as regulator of SR Ca(2+) uptake and Ca(2+) release. HRC might play a particularly relevant role in the equine heart, given its slower resting heart rate (20-35 beats/min) and longer action potential dura...
Circadian variation in ghrelin and certain stress hormones in crib-biting horses. Crib-biting is classified as an oral stereotypy, which may be initiated by stress susceptibility, management factors, genetic factors and gastrointestinal irritation. Ghrelin has been identified in the gastric mucosa and is involved in the control of food intake and reward, but its relationship to crib-biting is not yet known. The aim of this study was to examine the concentration and circadian variation of plasma ghrelin, cortisol, adrenocorticotropic hormone (ACTH) and β-endorphin in crib-biting horses and non-crib-biting controls. Plasma samples were collected every second hour for 24h in ...
Genetic differences in the serum proteome of horses, donkeys and mules are detectable by protein profiling. Although horses and donkeys belong to the same genus, their genetic characteristics probably result in specific proteomes and post-translational modifications (PTM) of proteins. Since PTM can alter protein properties, specific PTM may contribute to species-specific characteristics. Therefore, the aim of the present study was to analyse differences in serum protein profiles of horses and donkeys as well as mules, which combine the genetic backgrounds of both species. Additionally, changes in PTM of the protein transthyretin (TTR) were analysed. Serum protein profiles of each species (five anima...
Inhibition of cytochrome P450 enzymes involved in ketamine metabolism by use of liver microsomes and specific cytochrome P450 enzymes from horses, dogs, and humans. To identify and characterize cytochrome P450 enzymes (CYPs) responsible for the metabolism of racemic ketamine in 3 mammalian species in vitro by use of chemical inhibitors and antibodies. Methods: Human, canine, and equine liver microsomes and human single CYP3A4 and CYP2C9 and their canine orthologs. Methods: Chemical inhibitors selective for human CYP enzymes and anti-CYP antibodies were incubated with racemic ketamine and liver microsomes or specific CYPs. Ketamine N-demethylation to norketamine was determined via enantioselective capillary electrophoresis. Results: The general CYP inhibit...
Effects of in vivo lidocaine administration at the time of ischemia and reperfusion on in vitro contractility of equine jejunal smooth muscle. To determine whether administration of lidocaine during ischemia and reperfusion in horses results in concentrations in smooth muscle sufficient to protect against the negative consequences of ischemia-reperfusion injury on smooth muscle motility. Methods: 12 horses. Methods: Artificial ischemia and reperfusion injury of jejunal segments was induced in vivo in conjunction with lidocaine treatment during ischemia (IRL) or without lidocaine treatment (IR). Isometric force performance was measured in vitro in IRL and IR smooth muscle preparations with and without additional in vitro application o...
Ex vivo spontaneous generation of 19-norandrostenedione and nandrolone detected in equine plasma and urine. 19-Norandrostenedione (NAED) and nandrolone are anabolic-androgenic steroids (AASs). Nandrolone was regarded solely as a synthetic AAS until the 1980s when trace concentrations of apparently endogenous nandrolone were detected in urine samples obtained from intact male horses (stallions). Since then, its endogenous origin has been reported in boars and bulls; endogenous NAED and nandrolone have been identified in plasma and urine samples collected from stallions. More recently, however, it was suggested that NAED and nandrolone detected in urine samples from stallions are primarily artifacts d...
Equine pre-implantation conceptuses express neuraminidase 2–a potential mechanism for desialylation of the equine capsule. During the second and third week of pregnancy, the equine conceptus is covered by an acellular glycoprotein capsule. This capsule contains glycoproteins resembling those of the mucin family with sialic acid making up a high proportion of the carbohydrate. Coinciding with conceptus fixation, a marked decline in sialic acid content of the capsule occurs, which has been proposed to contribute to cessation of conceptus mobility. Herein, we describe the expression of neuraminidase 2 (NEU2) by pre-implantation stages of equine conceptus development. NEU2 transcript abundance was examined in conceptu...
Expression patterns of intestinal calcium transport factors and ex-vivo absorption of calcium in horses. In many species, the small intestine is the major site of calcium (Ca(2+)) absorption. The horse differs considerably from most other species with regard to the physiology of its Ca(2+) metabolism and digestion. Thus, this study was performed to get more information about the transcellular Ca(2+) absorption in the horse.Two mechanisms of intestinal Ca(2+) absorption are described: the passive paracellular pathway and the active, vitamin D-dependent transcellular pathway. The latter involves the following elements: vitamin D receptors (VDR), transient receptor potential vanilloid channel member...
Development and validation of a method for Cd, Pb and As analysis in bovine, equine and poultry liver by inductively coupled plasma mass spectrometry. A method for the determination of As, Cd and Pb in bovine, equine and poultry liver by ICP-MS was developed and validated. Samples were digested in a microwave oven using a 10% HNO(3) solution. A set of experiments was made according to a central composite design (CCD) for optimisation of the plasma argon flow, nebuliser argon flow and radiofrequency power applied to the plasma. During the validation, Rh and Ru were evaluated as internal standards and, after validation, the best was Rh for Pb and Cd analysis, but for As better results were obtained without an internal standard. The method allo...
Isolation and differentiation potential of an equine amnion-derived stromal cell line. Stem cells represent an important tool in veterinary therapeutic field such as tissue engineering. In the present study, equine amnion-derived mesenchymal stromal cells were investigated for applications in veterinary science as an alternative source to bone marrow mesenchymal stem cells and adipose stem cells. Amnion stromal cells isolation and characterization protocol is described; the in vitro cell growth rate was calculated by measuring viable cell number over 20 days. The expression of stem cell markers such as Oct-4, Nanog, Sox-2 and CD105 was assessed by retrotranscription quantitativ...
Quantitative analysis of a quaternary ammonium drug: ipratropium bromide by LC/ESI-MS(n) in horse plasma and urine. A quantitative method, using LC/ESI-MS(n) with a quadrupole linear ion trap mass analyzer, has been developed for the analysis of ipratropium cation in horse plasma and urine. The method applies solid-phase extraction with WCX cartridges for plasma and MM2 cartridges for urine, prior to analysis by LC/ESI-MS(n). The efficiency of extraction combined with the sensitivity and the selectivity of MS(n) allows for the quantification of ipratropium cation at picogram per milliliter levels. The analytical capabilities of the method have been successfully checked by the quantitative analysis of ipratr...
Comparison of cortisol and ACTH responses after administration of thyrotropin releasing hormone in normal horses and those with pituitary pars intermedia dysfunction. Changes in both adrenocorticotropin (ACTH) and cortisol concentration in response to thyrotropin releasing hormone (TRH) administration have been used to diagnose equine pituitary pars intermedia dysfunction (PPID), but the use of the 2 hormones has not been compared. Objective: Measuring ACTH concentration is superior to measuring cortisol concentration after TRH administration in differentiating between normal horses and those with PPID, and the 2 hormone concentrations are disassociated in PPID horses. Methods: Eleven horses and 2 ponies with PPID and 19 normal horses. Methods: A study eval...
Hormone concentration changes temporally associated with the hour of transition from preluteolysis to luteolysis in mares. The temporal associations of cortisol, estradiol-17β, and oxytocin with pulses of PGFM at the common hour of transition between preluteolysis and luteolysis was studied in plasma from hourly blood samples in mares (n=8). The transitional hour was determined from progesterone concentrations and occurred between 2PM and 2AM in all mares. Pulses of PGFM were grouped into those occurring at the last pulse of preluteolysis (preluteolytic pulse), at the hour of transition (transitional), and during luteolysis (luteolytic). The preluteolytic PGFM pulse (45±16pg/ml at peak) and transitional pulse (4...
Dose-response effects of estrogenic mycotoxins (zearalenone, alpha- and beta-zearalenol) on motility, hyperactivation and the acrosome reaction of stallion sperm. The aim of this study was to investigate the in vitro effects of the Fusarium fungus-derived mycotoxin, zearalenone and its derivatives alpha-zearalenol and beta-zearalenol on motility parameters and the acrosome reaction of stallion sperm. Since the toxic effects of zearalenone and its derivatives are thought to result from their structural similarity to 17beta-estradiol, 17beta-estradiol was used as a positive control for 'estrogen-like' effects. Methods: Stallion spermatozoa were exposed in vitro to zearalenone, alpha-zearalenol, beta-zearalenol or 17beta-estradiol at concentrations ranging...
Determination of sucrose in equine serum using liquid chromatography-mass spectrometry (LC/MS). Mucosal integrity may be objectively assessed by determination of the absorption of exogenous substances such as sucrose. Gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) have been reported for the accurate quantification of low concentrations of sucrose in serum. LC/MS offered the advantage of high sensitivity and mass selectivity without the need for extensive sample derivatization required for GC/MS methods. However, the high polarity and non-volatile nature of the sucrose molecule renders LC/MS techniques challenging. Previously published rep...
The effect of interval versus continuous exercise on plasma leptin and ghrelin concentration in young trotters. The effect of interval vs. continuous exercise on plasma leptin and ghrelin concentration in young Standardbred horses was studied. The experiment was conducted on 27 trotters, in the age between 2 and 3 years. They were divided into two groups according to the type of exercise. Blood samples were collected through jugular venipuncture in the following experimental conditions: at rest, immediately after exercise and 30 minutes after the end of the effort. Plasma leptin and ghrelin concentrations were determined using RIA tests. The continuous exercise induced an increase in plasma leptin conce...
Horse metabolism and the photocatalytic process as a tool to identify metabolic products formed from dopant substances: the case of sildenafil. Two horses were treated with sildenafil, and its metabolic products were sought in both urine and plasma samples. Prior to this, a simulative laboratory study had been done using a photocatalytic process, to identify all possible main and secondary transformation products, in a clean matrix; these were then sought in the biological samples. The transformation of sildenafil and the formation of intermediate products were evaluated adopting titanium dioxide as photocatalyst. Several products were formed and characterized using the HPLC/HRMS(n) technique. The main intermediates identified in thes...
Flow cytometric detection of myeloperoxidase in horse neutrophils: a novel technique in equine diagnostic research. Myeloperoxidase (MPO) is a protein of interest due to its involvement in equine pathologies. Until now, results in equine diagnostic research were achieved through extracellular MPO detection. However, studying the cellular MPO content in neutrophils has revealed important insights in human diseases. This study aimed to develop a technique for the specific detection of MPO on the single cell level defining a flow cytometric protocol for the detection of both equine surface-bound and cellular MPO. Both indirect and direct labeling techniques are described which include the comparison of two sec...
Oxidant-antioxidant imbalance in horses infected with equine infectious anaemia virus. This study assesses the impact of equine infectious anaemia virus (EIAV) infection on the oxidant/antioxidant equilibrium of horses. Blood samples from 96 Romanian horses aged 1-25 years, were divided into different groups according to their EIAV-infection status, age, and time post-seroconversion. The effect of infection on oxidative stress was estimated by measuring enzymatic antioxidants (superoxide dismutase [SOD], glutathione peroxidase [GPx] and catalase), non-enzymatic antioxidants (uric acid and carotenoids), and lipid peroxidation (malondialdehyde [MDA]). Infection modified the oxidan...
Hypoxia regulates the expression of extracellular matrix associated proteins in equine dermal fibroblasts via HIF1. Exuberant granulation tissue (EGT), a fibrotic healing disorder resembling the human keloid, occurs almost exclusively in limb wounds of horses and may be caused in part by a relative state of hypoxia within the wound. Objective: The objectives of this study were therefore to (1) assess the effects of hypoxia on equine dermal fibroblast (EDF) proliferation and apoptosis, (2) study the effects of hypoxia on the expression of key extracellular matrix (ECM) associated proteins and determine if such effects are dependent on hypoxia-inducible factor (HIF), and (3) determine if EDFs from the body or...
Urinary glycosaminoglycans in horse osteoarthritis. Effects of chondroitin sulfate and glucosamine. Our objectives were to characterize the urinary excretion of glycosaminoglycans (GAGs) in horse osteoarthritis, and to investigate the effects of chondroitin sulfate (CS) and glucosamine (GlcN) upon the disease. Urinary GAGs were measured in 47 athletic horses, 20 healthy and 27 with osteoarthritis. The effects of CS and GlcN were investigated in mild osteoarthritis. In comparison to normal, urinary GAGs were increased in osteoarthritis, including mild osteoarthritis affecting only one joint. Treatment with CS+GlcN led to a long lasting increase in the urinary CS and keratan sulfate (KS), and ...
A combination of single-drop microextraction and open tubular capillary electrochromatography with carbon nanotubes as stationary phase for the determination of low concentration of illicit drugs in horse urine. In this study we developed an interesting alternative to HPLC-mass spectrometry for the quantification of seven important drugs of abuse in racehorses. The procedure proposed in this work is a combination of single-drop microextraction (SDME) and an open tubular capillary electrochromatography (OT-CEC) using multi-wall carbon nanotubes (MWCTs) immobilized into a fused-silica capillary as a stationary phase. The SDME showed to be a powerful tool for extraction/preconcentration of the seven drugs analyzed in the study, showing an enrichment factor between 38- and 102-fold depending on the drug. ...
