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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.
Asymmetric histone 3 methylation pattern between paternal and maternal pronuclei in equine zygotes. Hoechst staining has traditionally been used to evaluate fertilization and parental origin of pronuclei. However, prevalence of parthenogenetic activation cannot be distinguished accurately by this protocol, and variation of relative pronuclear size and position makes it impossible to determine parental origin. We demonstrate that in equine zygotes, the epigenetic modification histone 3 lysine 9 trimethylation (H3K9me3) shows an asymmetric pattern between maternal and paternal pronuclei. H3K9me3 immunostaining appears to be a robust technique to identify the parent of origin of equine pronucle...
Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels. The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion...
Influence of stage of lactation and year season on composition of mares’ colostrum and milk and method and time of storage on vitamin C content in mares’ milk. Mares' milk is becoming increasingly popular in Western Europe. This study was thus aimed at investigating the impact of stage of lactation and season on chemical composition, somatic cell count and some physicochemical parameters of mares' colostrum and milk, and at developing a method for the determination of vitamin C (ascorbic acid) in mares' milk and to determine its content in fresh and stored milk. Results: The analysis conducted showed an effect of the stage of lactation on contents of selected chemical components and physicochemical parameters of mares' milk. In successive lactation p...
Orally supplemented L-arginine impairs amino acid absorption depending on dose in horses. The beneficial effect of L-arginine (L-Arg) supplementation, on the physiology of several species, has generated an interest in the use of L-Arg as a nutraceutical in horses, but dosage and absorption of orally supplemented L-Arg must be inferred from other species. The study objective was to determine the effect of 2 oral L-Arg doses on plasma arginine concentrations and the effect on absorption of other amino acids in mares. In Experiment 1, mares were blocked by age and breed and were fed L-Arg supplemented (supplemented with 0.025% BW L-Arg; n=6) or control (no supplement; n=6) concentrate...
Presence and function of dopamine transporter (DAT) in stallion sperm: dopamine modulates sperm motility and acrosomal integrity. Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility...
Oxytocin induction of pulses of a prostaglandin metabolite and luteolysis in mares. A procedure for oxytocin (OT) administration on Day 13 postovulation was developed in mares for stimulation of a pulse of PGFM (a PGF2α metabolite) that mimics a natural PGFM pulse during luteolysis. Bolus treatment with each of five OT doses (1-10 IU/mare, n = 3) stimulated a burst of PGFM that was maximum in 4 minutes and was unlike a natural pulse. A 2-hour OT infusion of 1.25, 2.5, or 5 IU/100 kg (n = 4) induced a PGFM pulse similar to reported pulses; lower doses did not. The peak of an induced pulse (approximately 260-380 pg/mL) seemed similar to reported natural peaks (approxima...
Hormonal, luteal, and follicular changes during initiation of persistent corpus luteum in mares. Mares with persistent CL (PCL) with no known etiology (idiopathic) were matched with mares with an interovulatory interval (IOI) of apparent physiological length, so that ovulation at the beginning of each PCL and IOI occurred during the same month (n = 6/group). Blood samples were collected daily from Days 12 to 22 (Day 0 = ovulation). Mean progesterone (P4) decreased in both groups on Days 14 and 15 and then diverged with a continued decrease in the IOI group and the beginning of constant and greater (P < 0.05) P4 concentration on each day in the PCL group. Before P4 divergence betw...
Purification of equine chorionic gonadotropin (eCG) using magnetic ion exchange adsorbents in combination with high-gradient magnetic separation. Current purification of the glycoprotein equine chorionic gonadotropin (eCG) from horse serum includes consecutive precipitation steps beginning with metaphosphoric acid pH fractionation, two ethanol precipitation steps, and dialysis followed by a numerous of fixed-bed chromatography steps up to the specific activity required. A promising procedure for a more economic purification procedure represents a simplified precipitation process requiring only onethird of the solvent, followed by the usage of magnetic ion exchange adsorbents employed together with a newly designed 'rotor-stator' type Hi...
Preliminary investigation into a potential role for myostatin and its receptor (ActRIIB) in lean and obese horses and ponies. Obesity is a widespread problem across the leisure population of horses and ponies in industrialised nations. Skeletal muscle is a major contributor to whole body resting energy requirements and communicates with other tissues through the secretion of myokines into the circulation. Myostatin, a myokine and negative regulator of skeletal muscle mass, has been implicated in obesity development in other species. This study evaluated gene and protein expression of myostatin and its receptor, ActRIIB in adipose tissues and skeletal muscles and serum myostatin concentrations in six lean and six obes...
Calcium-sensing receptor-mediated osteogenic and early-stage neurogenic differentiation in umbilical cord matrix mesenchymal stem cells from a large animal model. Umbilical cord matrix mesenchymal stem cells (UCM-MSCs) present a wide range of potential therapeutical applications. The extracellular calcium-sensing receptor (CaSR) regulates physiological and pathological processes. We investigated, in a large animal model, the involvement of CaSR in triggering osteogenic and neurogenic differentiation of two size-sieved UCM-MSC lines, by using AMG641, a novel potent research calcimimetic acting as CaSR agonist. Results: Large (>8 µm in diameter) and small (<8 µm) equine UCM-MSC lines were cultured in medium with high calcium (Ca2+) concentration (...
Enzymatic scavengers in the epididymal fluid: comparison between pony and miniature breed stallions. The use of stallion semen collected from cauda epididymis for AI has increased due to the new protocols available for cryopreservation. Preserving the genetic material from valuable males that suffer sudden death or other events that prematurely end the stallion's reproductive life is an important strategy for Stud breeding management. While protecting spermatozoa from oxidative stress and infectious agents, the epididymis promotes the enhancement of sperm cell morphology and changes in membrane protein profile, increasing its fertility potential. The epididymal fluid must be a balanced redox ...
Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: presence and sensitivity of 5′ AMP-activated protein kinase (AMPK). This study evaluated the effect of the use of hypometabolic TRIS extenders in the presence or the absence of AMPK activators as well as the utilization of high cooling rates in the refrigeration step on the freezability of stallion sperm. Twelve ejaculates were cryopreserved using Botucrio® as a control extender and a basic TRIS extender (HM-0) separately supplemented with 10 mM metformin, 2mM 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), 2 mM Adenosine monophosphate (AMP), 40 μM compound C AMPK inhibitor or 2 mM AMP+40 μM compound C. Our results showed that the utilization ...
Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI. The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses ...
Anti-loxoscelic horse serum produced against a recombinant dermonecrotic protein of Brazilian Loxosceles intermedia spider neutralize lethal effects of Loxosceles laeta venom from Peru. In this work, an anti-loxoscelic serum was produced by immunizing horses with a recombinant dermonecrotic protein from Loxosceles intermedia (rLiD1). Anti-rLiD1 antibodies were able to recognize different species of Loxosceles venoms by Western Blot and ELISA. The efficacy of anti-rLiD1 serum against the toxic effects of Loxosceles laeta (Peru) venom was tested, showing that anti-rLiD1 serum can neutralize those effects. This study confirms that recombinant proteins can be good candidates to replace crude venoms for antivenom production.
Repeated measurements of P retention in ponies fed rations with various Ca:P ratios. This study addresses the question of whether feeding rations rich in P for a period of up to 42 d induces a positive P balance in adult ponies. Biochemical bone markers and parathyroid hormone (PTH; intact as well as whole PTH) were measured to obtain clues as to the effect of P loading on bone metabolism. The experiment had a Latin square design. Each feeding period lasted 42 d, and there were 2 balance trials (ECP1 and ECP2) within each feeding period. Each balance trial lasted 10 d (ECP1: d 11 to 21; ECP2: d 33 to 42). Six ponies aged 2.5 to 7 yr were fed a control diet that provided P and ...
Evaluation of ultrasmall superparamagnetic iron oxide contrast agent labeling of equine cord blood and bone marrow mesenchymal stromal cells. To evaluate the efficacy and effects of labeling equine umbilical cord blood (UCB)- and bone marrow (BM)-derived multipotent mesenchymal stromal cells (MSCs) with an ultrasmall superparamagnetic iron oxide (SPIO) contrast agent and the detection of labeled MSCs by use of MRI. Methods: UCB MSCs from placental tissues of 5 foals and BM MSCs from 5 horses. Methods: UCB and BM MSC cultures were seeded in duplicate (5,000 cells/cm(2)). One duplicate was incubated with SPIO (50 μg/mL); the other was processed identically, but without SPIO. Mesenchymal stromal cells were expanded in triplicates for ...
Andrographolide Exerts Chondroprotective Activity in Equine Cartilage Explant and Suppresses Interleukin-1 β -Induced MMP-2 Expression in Equine Chondrocyte Culture. Cartilage erosion in degenerative joint diseases leads to lameness in affected horses. It has been reported that andrographolide from Andrographis paniculata inhibited cartilage matrix-degrading enzymes. This study aimed to explore whether this compound protects equine cartilage degradation in the explant culture model and to determine its effect on matrix metalloproteinase-2 (MMP-2) expression, a matrix-degrading enzyme, in equine chondrocyte culture. Equine articular cartilage explant culture was induced by 25 ng/mL interleukin-1β, a key inducer of cartilage degeneration, in cultures with...
Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm. Repeatable methods for IVF have not been established in the horse, reflecting the failure of standard capacitating media to induce changes required for fertilization capacity in equine sperm. One important step in capacitation is membrane cholesterol efflux, which in other species is triggered by cholesterol oxidation and is typically enhanced using albumin as a sterol acceptor. We incubated equine sperm in the presence of calcium, BSA, and bicarbonate, alone or in combination. Bicarbonate induced an increase in reactive oxygen species (ROS) that was abolished by the addition of calcium or BSA...
Regulation of axonemal motility in demembranated equine sperm. Equine in vitro fertilization is not yet successful because equine sperm do not effectively capacitate in vitro. Results of previous studies suggest that this may be due to failure of induction of hyperactivated motility in equine sperm under standard capacitating conditions. To evaluate factors directly affecting axonemal motility in equine sperm, we developed a demembranated sperm model and analyzed motility parameters in this model under different conditions using computer-assisted sperm analysis. Treatment of ejaculated equine sperm with 0.02% Triton X-100 for 30 sec maximized both permeab...
Expression and regulation of regulator of G-protein signaling protein-2 (RGS2) in equine and bovine follicles prior to ovulation: molecular characterization of RGS2 transactivation in bovine granulosa cells. The luteinizing hormone preovulatory surge stimulates several signal pathways essential for ovulation, and the regulator of G-protein signaling protein-2 (RGS2) is thought to be involved in this process. The objectives of this study were to characterize the regulation of RGS2 transcripts in equine and bovine follicles prior to ovulation and to determine its transcriptional control in bovine granulosa cells. To assess the regulation of equine RGS2 prior to ovulation, RT-PCR was performed using total RNA extracted from equine follicles collected at various times after human chorionic gonadotropi...
Upflow anaerobic solid-state (UASS) digestion of horse manure: Thermophilic vs. mesophilic performance. Energetic use of complex lignocellulosic wastes has gained global interest. Thermophilic digestion of horse manure based on straw was investigated using the upflow anaerobic solid-state (UASS) process. Increasing the organic loading rate from 2.5 to 5.5gvsL(-)(1)d(-)(1) enhanced the average methane production rate from 0.387 to 0.687LCH4L(-)(1)d(-)(1), whereas the yield decreased from 154.8 to 124.8LCH4kgvs(-)(1). A single-stage and two-stage process design showed almost the same performance. Compared to prior experiments at mesophilic conditions, thermophilic conditions showed a significantly...
Hormonal composition of follicular fluid from abnormal follicular structures in mares. The objective was to characterise the hormonal composition of follicular fluid from mares with distinct anovulatory-cystic follicles. Follicular fluid was aspirated from six mares that presented with cystic follicles and from pre-ovulatory follicles of five normal mares (controls). Differences in progesterone, oestradiol, testosterone, IGF-I and IGF binding were analysed using Fisher's exact test. There were greater (P < 0.03) follicular fluid oestradiol concentrations in normal follicles and the testosterone concentration of the cystic fluid was greater (P < 0.05) than that of t...
Leptin and leptin receptor are detectable in equine spermatozoa but are not involved in in vitro fertilisation. In human and swine, leptin (OB) has been identified in seminal plasma and leptin receptors (OB-R) on the cell surface of spermatozoa, indicating that spermatozoa are a target for OB. This hormone has also been detected in follicular fluid (FF) in women and mares, although its role requires further study. The aims of this study were to investigate the immunolocalisation and the expression of OB and OB-R in equine spermatozoa and to evaluate the involvement of OB in equine in vitro fertilisation (IVF). Since progesterone (P) and OB are both found in FF, the individual and combined effects of the...
Peripheral Blood-Derived Mesenchymal Stromal Cells Promote Angiogenesis via Paracrine Stimulation of Vascular Endothelial Growth Factor Secretion in the Equine Model. Mesenchymal stromal cells (MSCs) have received much attention as a potential treatment of ischemic diseases, including ischemic tissue injury and cardiac failure. The beneficial effects of MSCs are thought to be mediated by their ability to provide proangiogenic factors, creating a favorable microenvironment that results in neovascularization and tissue regeneration. To study this in more detail and to explore the potential of the horse as a valuable translational model, the objectives of the present study were to examine the presence of angiogenic stimulating factors in the conditioned medium...
Progress on low susceptibility mechanisms of transmissible spongiform encephalopathies. Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are a group of fatal neurodegenerative diseases detected in a wide range of mammalian species. The "protein-only" hypothesis of TSE suggests that prions are transmissible particles devoid of nucleic acid and the primary pathogenic event is thought to be the conversion of cellular prion protein (PrP(C)) into the disease-associated isoform (PrP(Sc)). According to susceptibility to TSEs, animals can be classified into susceptible species and low susceptibility species. In this review we focus on several species with l...
Gene and protein expression and cellular localisation of cytochrome P450 enzymes of the 1A, 2A, 2C, 2D and 2E subfamilies in equine intestine and liver. Among the cytochrome P450 enzymes (CYP), families 1-3 constitute almost half of total CYPs in mammals and play a central role in metabolism of a wide range of pharmaceuticals. This study investigated gene and protein expression and cellular localisation of CYP1A, CYP2A, CYP2C, CYP2D and CYP2E in equine intestine and liver. Real-time polymerase chain reaction (RT-PCR) was used to analyse gene expression, western blot to examine protein expression and immunohistochemical analyses to investigate cellular localisation. Results: CYP1A and CYP2C were the CYPs with the highest gene expression in the ...
Cryotolerance of stallion spermatozoa is related to ROS production and mitochondrial membrane potential rather than to the integrity of sperm nucleus. Although cryopreservation of stallion spermatozoa allows long-term preservation of spermatozoa from particular stallions and facilitates international trade, it is understood to inflict damages on sperm cells that may finally reduce their fertilizing ability. In addition, individual differences are known to exist in the sperm ability to withstand freeze-thawing protocols. To date, these differences have mainly been reported on the basis of sperm motility and membrane integrity. For this reason, the present work sought to determine differences between good (good freezability ejaculates: GFE) an...
Development of a method for absolute quantification of equine acute phase proteins using concatenated peptide standards and selected reaction monitoring. The aim of this study was the development of a quantitative assay that could support future studies of a panel of acute phase proteins (APPs) in the horse. The assay was based on a quantification concatamer (QconCAT) coupled to selected reaction monitoring methodology. Thirty-two peptides, corresponding to 13 putative or confirmed APPs for the Equus caballus (equine) species were selected for the design of a QconCAT construct. The gene encoding the QconCAT was synthesized and expressed as an isotope-labeled chimaeric protein in Escherichia coli. The QconCAT tryptic peptides were analyzed on a ...
The effects of sample handling and N-phenylmaleimide on concentration of adrenocorticotrophic hormone in equine plasma. Previous reports suggest that adrenocorticotrophic hormone (ACTH) degrades rapidly, limiting its use as a diagnostic test. Objective: This study quantified effects of processing delays on ACTH concentrations and investigated the addition of N-phenylmaleimide (maleimide), a protease inhibitor, as a means of reducing ACTH degradation. Methods: Experimental study. Methods: Venous blood was collected from 8 healthy horses and 8 horses with pituitary pars intermedia dysfunction (PPID) with a range of ACTH concentrations. Baseline ACTH concentrations were established immediately using a chemilumines...
Effects of the combination of microfracture and self-assembling Peptide filling on the repair of a clinically relevant trochlear defect in an equine model. The goal of this study was to test the ability of an injectable self-assembling peptide (KLD) hydrogel, with or without microfracture, to augment articular cartilage defect repair in an equine cartilage defect model involving strenuous exercise. Methods: Defects 15 mm in diameter were created on the medial trochlear ridge and debrided down to the subchondral bone. Four treatment groups (n = 8 each) were tested: no treatment (empty defect), only defect filling with KLD, only microfracture, and microfracture followed by filling with KLD. Horses were given strenuous exercise throughout the one-ye...
