Topic:Laboratory Methods
Laboratory methods in equine research encompass a variety of techniques and procedures used to analyze biological samples from horses to study health, disease, genetics, and physiology. These methods include hematological analyses, biochemical assays, molecular biology techniques, and microbiological cultures. Commonly utilized laboratory techniques involve blood tests for complete blood count (CBC) and serum chemistry, polymerase chain reaction (PCR) for genetic and infectious disease studies, and enzyme-linked immunosorbent assays (ELISA) for detecting specific proteins or antibodies. These methods provide valuable data that contribute to understanding equine health and disease mechanisms. This page compiles peer-reviewed research studies and scholarly articles that explore the application, development, and outcomes of laboratory methods in the context of equine research.
Retrospective Evaluation of Synchronous Diaphragmatic Flutter in Horses: 37 Cases (2004-2022). To describe the clinical presentation of horses with synchronous diaphragmatic flutter (SDF) and identify clinical and laboratory variables associated with survival. Methods: Retrospective study from January 2004 to September 2022. Methods: Five large animal referral hospitals in the United States. Methods: Thirty-seven horses of various breeds and ages presenting with clinical signs of SDF, excluding animals participating in an endurance competition. All horses were >1Â year of age. Methods: None. Results: Variables recorded and analyzed included signalment, clinical and laboratory data, trea...
Quantitative modeling reveals prolonged lactate clearance in young and unraced Jeju horses following high-intensity exercise under passive recovery conditions. To model lactate recovery kinetics in relation to sex, age, running speed, and racing experience, and to estimate blood lactate clearance time under passive recovery conditions that reflect current management practices in Jeju horses, a less well-characterized pony-sized indigenous breed. Unassigned: From October through December 2022, BLC was measured at baseline, 10 minutes after 800-m barrier trials (time [T]-1), and 50 minutes (T2) after 800-m barrier trials in 40 race-trained Jeju horses (2 to 4 years old). Lactate clearance was modeled using nonlinear regression with an exponential decay...
A Markerless Approach for Full-Body Biomechanics of Horses. The ability to quantify equine kinematics is essential for clinical evaluation, research, and performance feedback. However, current methods are challenging to implement. This study presents a motion capture methodology for horses, where three-dimensional, full-body kinematics are calculated without instrumentation on the animal, offering a more scalable and labor-efficient approach when compared with traditional techniques. Kinematic trajectories are calculated from multi-camera video data. First, a neural network identifies skeletal landmarks (markers) in each camera view and the 3D location...
α2-Macroglobulin Concentrations in Canine and Equine Serum Donors. To determine if serum α2-macroglobulin (A2M) concentration varies based on donor species-canine or equine-and signalment. Methods: Serum A2M concentration ([A2M]) was measured in healthy dogs (n = 30) and horses (n = 31) using species-specific ELISAs. Results: Canine and equine [A2M] median (IQR; range) were 98.70 ng/mL (92.79 ng/mL; 34.33-696.18 ng/mL) and 557 000 ng/mL (437 900 ng/mL; 62 600-3 042 900 ng/mL), respectively. Equine [A2M] was significantly higher than canine [A2M]. Depending on the statistical analysis performed, sex had either no statistical (pâ€...
Seasonal elevation in equine adrenocorticotropic hormone occurs throughout the contiguous United States and is influenced by latitude and age. To determine whether seasonal variation in equine plasma ACTH concentrations is affected by latitude between 29.7°N and 45.0°N in the US. Unassigned: A cohort study was performed between June 2019 and May 2020 in which plasma ACTH was measured monthly by chemiluminescence immunoassay. The study involved 201 apparently healthy horses from 9 locations within the US classified into northern, central, and southern latitude cohorts. Data analysis was performed with 3 major objectives: (1) identify outlier ACTH concentrations and horses; (2) estimate ACTH upper reference limits to define expected ...
Blastocyst production by conventional in vitro fertilization (cIVF) in horses: Effects of sperm storage method, incubation timing of cool-stored semen before gamete co-incubation, and comparisons between cIVF and intracytoplasmic sperm injection (ICSI). Currently, Intracytoplasmic Sperm Injection (ICSI) on in vitro-matured equine oocytes obtained by transvaginal oocyte aspiration (TVA) is the method of choice for in vitro production of equine blastocysts in a clinical setting. A protocol for conventional in vitro fertilization (cIVF) using either fresh or frozen/thawed stallion sperm incubated under capacitating conditions (Tyrode's Albumin Lactate Pyruvate medium + penicillamine, hypotaurine, epinephrine; FERT-PHE) has been recently reported. Several factors that may impact the clinical applicability of this cIVF protocol have yet to be st...
Histopathological and molecular evaluation of hydatid cysts according to necropsy findings of 52 equids in Türkiye. is a parasitic cestode primarily maintained within an equine-canine life cycle, with horses, donkeys, mules, and other ungulates serving as intermediate hosts. Although has historically been considered non-zoonotic, recent molecular studies suggest that this assumption may need to be reevaluated. This study aimed to investigate the presence and molecular characterization of in equids from Türkiye. A retrospective analysis of 52 equine necropsies performed between 2020 and 2025 identified hydatid cysts in one Arabian horse and two donkeys. Gross and histopathological examination confirmed t...
Using an Equine Cadaver Head to Investigate Associations Between Sub-Noseband Space, Noseband Tension, and Sub-Noseband Pressure at Three Locations. Pressures applied to horses via nosebands are of growing concern. The current study applied noseband pressure to the head of a dead horse. Pressure sensors were placed on the left nasal bone to record pressures as the noseband was progressively tightened. Tightness increased as predicated by holes in the strap of the noseband (as supplied) through eight steps from two fingers' space, assessed using the standard International Society for Equitation Science Taper Gauge through to zero space. Sensors were also placed at the midline frontal plane and intra-orally at the level of the second premola...
The magnitude of hyperchloremia in horses is associated with diarrhea and renal function. To describe the signalment, diagnosis, treatment, and laboratory characteristics of horses with hyperchloremia and identify factors associated with the magnitude of the increased chloride concentration. Unassigned: Horses with a chloride concentration greater than 108 mEq/L were included in this retrospective study. Descriptive statistics and multiple linear regression analysis were used to identify associations between diagnosis, treatment, and laboratory variables and the magnitude of hyperchloremia. Unassigned: 134 horses met the inclusion criteria for this study. Creatinine was a significa...
Reference Intervals for Biochemical Analytes in Clinically Healthy Adult Lusitano Horses. To date, serum biochemical analytes reference intervals (RIs) in Lusitano horses have not been studied. This study aimed to establish the RIs for biochemical analytes following the American Society of Veterinary Clinical Pathology guidelines and to compare them with the general equine population's RIs. Blood samples were collected from 76 clinically healthy adult Lusitano horses, and RIs of 22 biochemical variables were determined using Reference Value Advisor software. Lusitano horse-specific RIs are proposed for the following variables: total protein (3.9-7.0 g/dL), albumin (2.5-3.8 g/dL), g...
Gray-Horse Melanoma-A Wolf in Sheep’s Clothing. Malignant melanoma (MM) affects not only humans but also animals, with gray horses being particularly predisposed to acquiring the disease. Multiomics have greatly advanced the understanding of human MM. In contrasty little is known regarding the pathogenesis of gray-horse melanoma and the unique phenomenon of melanoma "dormancy" in some animals. To help close this gap in knowledge, melanoma tissue and intact skin collected from gray horses were subjected to transcriptome analysis using RNAseq. In the next step, cultured primary tumor cells and normal skin fibroblasts were established from gra...
Phylogenetic variation of layer II cortical immature neurons in dog and horse confirms covariance with brain size and neocortical surface. Recent research in brain structural plasticity has identified "immature" or "dormant" neurons in layer II of the cerebral cortex (cortical immature neurons; cINs), cells that remain in a prolonged state of arrested development but retain the ability to resume maturation and integrate functionally into mature cortical circuits. These immature cells are far more abundant in large-brained mammals, being restricted to paleocortex (piriform cortex) in small-brained rodents and extending in the widely expanded neocortical mantle of species with large gyrencephalic brains. In a previous systematic an...
Evaluation of Blood Biochemical Parameters and Ratios in Piroplasmosis-Infected Horses in an Endemic Region. Equine piroplasmosis (EP), caused by and , is a worldwide tick-borne disease with severe economic, commercial, and sanitary implications for equids. Although diagnosis is based on direct (blood smear or PCR) or indirect (serology) methods, these techniques are expensive, laborious, and false-negative and false-positive results can be yielded. Biochemistry blood profiles are routinely performed in horses. Biochemical parameters and ratios could be a reliable complementary diagnostic tool to assist clinicians in EP diagnosis, mainly in endemic areas, or for discarding similar disorders (piro-li...
Comprehensive Integrated Analyses of Proteins and Metabolites in Equine Seminal Plasma (Horses and Donkeys). The reproductive ability of equine species is a critical component of equine breeding programs, with sperm quality serving as a primary determinant of reproductive success. In this study, we perform an integrative analysis of proteomics and metabolomics in seminal plasma to identify proteins and metabolites associated with sperm quality and reproductive ability in equine species. Methods: We utilized the CEROS instrument to assess the morphology and motility of sperm samples from three horses and three donkeys. Additionally, we statistically analyzed the mating frequency and pregnancy rates in...
Effect of Pre-IVM Duration with cAMP Modulators on the Production of Cloned Equine Embryos and Foals. The asynchrony of cytoplasmic and nuclear maturation in cumulus-oocyte complexes (COCs) due to prematurely declining concentrations of cyclic adenosine monophosphate (cAMP) has been shown to result in reduced oocyte developmental competence. The objective of this study was to evaluate the effect of pre-IVM treatment with cAMP modulators for different durations on the developmental potential of equine oocytes used for cloned embryo production. Collected COCs were transferred to cryovials filled with transport medium at 20-22 °C. Within the cryovials, the COCs were either untreated (Control) fo...
Direct Warming of Vitrified In Vivo Equine Embryos. Vitrified in vitro-produced embryos can be successfully warmed in isotonic media at room temperature (RT; 22°C). However, this protocol has not been reported for in vivo embryos, which are more challenging to vitrify and warm. Study objectives were to see if vitrified in vivo embryos warmed in RT isotonic medium gave equivalent pregnancy rates to stepwise serial dilution warming, and if embryo size influenced the results. One hundred and seventeen embryos were divided into groups by size (G1:≤ 300 μm, n = 59; G2:> 300-400 μm, n = 33; G3:> 400-500 μm, n = ...
Analysis of Motion Characteristics and Plasma Membrane Intactness (Viability) in Sperm from Domestic Animals. Sperm quality analysis using computer-assisted sperm analysis (CASA) systems and fluorescence-based techniques has become common in the animal reproduction industry, particularly for large animals (i.e., bovine, porcine, equine). In this chapter, the methods commonly utilized in the author's laboratory to examine sperm motion characteristics via CASA and plasma membrane intactness by flow cytometry will be described. These include methods to properly dilute fresh (stallions, bulls, boars), cool-stored (stallions, boars), or frozen/thawed (stallions, bulls, boars) sperm for assessment of sperm ...
Quantitative proteomics unveils potential plasma biomarkers and provides insights into the pathophysiological mechanisms underlying equine metabolic syndrome. Equine Metabolic Syndrome (EMS) is a multifactorial endocrine disorder characterized by obesity, insulin dysregulation (ID), and an increase in the risk of laminitis, a painful condition that can lead to euthanasia in severe cases. Diagnosing EMS is challenging and often relies on clinical history including obesity, difficulty in losing weight, and recurring episodes of laminitis. The gold standard for laboratory support of an EMS diagnosis is the identification of ID, with basal insulin being the simplest and most accessible method, especially in a field setting. However, various factors such...
Transcriptional Factors Related to Cellular Kinetics, Apoptosis, and Tumorigenicity in Equine Adipose-Derived Mesenchymal Stem Cells (ASCs) Are Influenced by the Age of the Donors. The impact of donor age on Adipose-derived mesenchymal stem cell (ASC) functionality and safety remains insufficiently characterized, particularly in equine models. This study investigates the influence of age on ASCs proliferation dynamics and the expression of tumorigenic and apoptosis-related markers. Equine ASCs were isolated from juvenile (<5 years), middle-aged (5-15 years), and geriatric (>15 years) horses and assayed across multiple passages. The relative mRNA expressions of pluripotency (Oct4), tumorigenic (CA9), and apoptosis-related (Bax and Bcl 2) markers were evaluated. The Gomper...
Influence of Cryopreservation on the Acrosome Reaction in Hucul Stallion Spermatozoa. The Hucul horse is a Polish primitive breed with a small population size, which highlights the importance of preserving the genetic resources. The cryopreservation of semen is essential for creating gene banks, but its effect on the acrosome reaction in Hucul stallions has not yet been investigated. The acrosome reaction is one of the most important physiological events associated with the fertilization process. Therefore, our goal was to determine the level of acrosome reaction in chilled and frozen/thawed Hucul stallion semen using the FluoAcro test and the SCA semen analysis system. We foun...
A comparison of the efficacy of three commercial human embryo vitrification kits for cryopreservation of in vivo produced equine embryos. Different cryoprotectants can influence the ability of embryos to successfully survive vitrification and subsequent warming before transfer. Objective: To compare pregnancy rates for embryos ≤500 μm vitrified, without puncture or aspiration of the blastocoele cavity, with one of three commercial human embryo vitrification kits containing the same penetrating cryoprotectants (DMSO and EG) but varying in their non-penetrating cryoprotectants (NPCPAs; sucrose, trehalose, dextran serum supplement [DSS], and hydroxypropyl cellulose [HPC]). Methods: In vivo experiments. Methods: Embryos (n =â...
Haemoglobin concentration measurement agreement obtained from three different devices in anaesthetised horses. Accurate measurement of haemoglobin concentration is crucial for monitoring the oxygen-carrying capacity in anaesthetised horses. This prospective study aimed to determine the agreement in haemoglobin concentration in anaesthetised horses using the SpHb pulse co-oximeter, an Epoc® blood gas analyser, and values derived from packed cell volume (PCV), which served as the reference method in this study. The study included 34 horses undergoing elective surgeries. Blood samples were collected from each horse and divided for analysis using the microhaematocrit method to estimate haemoglobin concent...
Detection using chamber digital PCR with a DNA extraction-free method for gene-doping control. Gene doping, which entails the administration of transgenes, poses a serious threat to the integrity of equine sports and also raises both ethical and regulatory concerns. Current methods used for the detection of such doping often necessitate the extraction of DNA from plasma, which can be time-consuming and labour-intensive. To overcome this limitation, we developed a direct chamber digital PCR (cdPCR) method that enables transgene detection in equine plasma without the need for DNA purification. Using the equine erythropoietin (EPO) transgene as a model, we validated the assay by analysing ...
Effect of Centrifugation of Stallion Semen Through a Low Density Colloid Prior to Freezing on Sperm Cryosurvival. Sperm quality is adversely affected by cryopreservation due to the increased production of reactive oxygen species, which affects the integrity of sperm membranes, motility, and DNA fragmentation. Three methods for removing seminal plasma, washing (centrifuging extended semen at 800× for 10 min) and Single Layer Centrifugation with high or low density Equicoll, were used to prepare 29 ejaculates from ten stallions for freezing. Sperm quality parameters (kinematics, plasma membrane integrity, superoxide and hydrogen peroxide production, mitochondrial membrane potential, and DNA fragmentation)...
Analytical Data Review on an Artificial Intelligence Platform for Doping Control in Horse Racing. In the screening of prohibited substances (PS) in horse biological samples with gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) for doping control, an enormous number of chromatograms are generated. Reviewing these chromatograms to identify suspicious findings requires an extensive manual effort. Recent advancements in Artificial Intelligence (AI) enable its use to classify images into different categories. This can potentially be utilized to perform first-line analysis of chromatograms, which are usually displayed as images, by classifying them...
Detection of Transdermal Application of Testosterone to Racehorses by Analysis of Urine and Plasma. The use of testosterone in racehorses is predominantly monitored using international urine and plasma concentration-based thresholds and complementary steroid ratios. To date, there has been no published pharmacokinetic study on transdermally applied testosterone products in horses and whether their use could result in adverse analytical findings. Therefore, quantitative analysis of testosterone and epitestosterone in urine and testosterone in plasma samples was performed following a pilot multi-dose transdermal Testogel administration (1 mg/kg once a day for 7 days on clipped skin) to one...
Isolation of SSEA-3-positive mesenchymal stem cells from equine bone marrow and evaluation of their pluripotency. Generally, most mesenchymal stem cells (MSCs) have lower pluripotency and limited differentiation potential than embryonic stem cells (ESCs). However, a small subpopulation of MSCs, called multilineage differentiating stress-enduring (MUSE) cells, exhibit pluripotency. MUSE cells express stage-specific embryonic antigen 3 (SSEA-3), a sphingoglycolipid. Here, we isolated and investigated the pluripotency of SSEA-3-positive MSCs (MUSE cells). Six thoroughbred horses were used as test subjects. MSCs were harvested from the bone marrow of the thoracic vertebrae under ultrasound guidance. Harvested...
Advancements in microfluidic and electrophoretic techniques for stallion sperm isolation. Context Equine reproductive technologies are crucial for overcoming challenges in natural fertilisation, particularly in sub-fertile stallions and breeding programs focused on genetic conservation and performance enhancement. Assisted reproductive technologies (ARTs), such as artificial insemination (AI), intracytoplasmic sperm injection (ICSI), and in vitro fertilisation (IVF), improve fertility outcomes and enable breeding across geographical distances. Aims This review examines sperm isolation techniques used in ART, evaluating their efficacy, limitations, and potential to enhance reproduct...
Isolation and Characterization of Equine Mesenchymal Stromal/Stem Cells. Regenerative medicine is a relatively new branch of therapeutics in equine medicine, which aims to restore and reconstitute tissue function and structure via cellular and/or noncellular approaches. Biological constituents such as mesenchymal stromal/stem cells (MSCs) are potent therapeutics, which can aid in damaged tissue regeneration due to their differentiation capacity into many different cell types such as adipose tissue, bone, and cartilage. MSCs can be successfully and conveniently isolated from equine subcutaneous adipose tissue (adipose-derived stromal cells, ASCs). In horses, there a...
Knowledge levels regarding antibiotics and their use among horse owners in the State of Rio Grande do Norte, Brazil. This study aimed to evaluate the use of antimicrobials in horses, focusing on administration practices, adherence to veterinary prescriptions, and the impact on the development of antimicrobial resistance. The research was based on a questionnaire completed by 220 horse owners in the State of Rio Grande do Norte, Brazil. The data collected covered aspects including antibiotic use, prescription sources, the performance of culture and susceptibility tests, and the disposal of veterinary drug waste. The results indicated that 98.63% of horse owners administered antibiotics mainly to treat respira...