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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.
Reduced centrifugation duration improves diagnostic performance of an equine tapeworm egg counting technique. Anoplocephala perfoliata, can cause severe health problems in horses. Standard coprological tests have performed poorly for detecting A. perfoliata, and while a centrifugation-enhanced flotation technique is superior to other techniques, it is time consuming due to three 10-minute centrifugation steps. This study aimed to evaluate modifications of this technique that reduce the centrifugation durations to 5 and 1 min, respectively. Fecal samples were collected from ten adult A. perfoliata egg count positive mares and divided into 9 subsamples to allow for three replicate counts for each of th...
OPTIMIZING EXTRACTION AND CONCENTRATION METHODS FOR LC-MS/MS ANALYSIS OF VETERINARY PHARMACEUTICALS IN EQUINE-ASSOCIATED ENVIRONMENTAL MATRICES. This study developed a method for the simultaneous quantification of six potentially environmentally toxic veterinary drugs: benzylpenicillin, acepromazine, flunixin, dexamethasone, ketamine and romifidine, in soils, sediments, equine faeces and natural waters. Three methods were compared for their extraction from solid matrices: Ultrasonic extraction (USE), QuEChERS and Accelerated Solvent Extraction (ASE). ASE method was optimized according to temperature and extraction cycles. Quantification was performed by LC-MS/MS. LC and MS parameters were optimized. The method was validated according t...
Ovum pick-up in mares: Evidence review and best practice recommendations using a modified GRADE approach. Ovum pick-up (OPU) is a procedure that is now performed in mares worldwide to harvest oocytes for in vitro embryo production. There is a lack of consensus surrounding best practices for various procedural elements of the technique and recently there has been an increasing focus on the welfare aspects of the procedure. Our objective was to summarize current practice and to develop guidelines for OPU in the mare, using a modified GRADE approach to optimize efficiency and mare welfare. Published evidence on various aspects of the procedure was compiled, and to supplement the relative lack of publ...
Evaluation of two point-of-care molecular diagnostic platforms for rapid detection of equine Hendra virus. Hendra virus (HeV) is a lethal zoonotic pathogen endemic to eastern Australia, posing significant risks to equine and human health. Rapid field detection of HeV enables timely intervention and outbreak management. This study evaluated candidate point-of-care (POC) molecular diagnostic platforms for HeV detection in equine samples: including a loop-mediated isothermal amplification (DARQ RT-LAMP) assay and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). Comparative analytical evaluation demonstrated that RT-qPCR exhibited superior analytical sensitivity relativ...
Water and cryoprotectant permeability of mature equine oocytes: experimental measurements and in silico predictions. Vitrification of equine oocytes is an essential practice for advancing assisted reproductive technologies however, its efficiency remains limited due to the lack of stage and species-specific information on membrane permeability parameters. In this study, water (L) and CPA permeability (P) for dimethyl sulfoxide (Me₂SO) and ethylene glycol (EG) were measured in in vitro matured (MII) equine oocytes. Cumulus oocyte complexes were obtained from abattoir ovaries or by ovum pick-up and matured in vitro for 30 h at 6% CO. Oocytes followed ideal osmometer behavior principles, with an osmotically ...
Equine Liver Histopathology. This review summarizes best practices for equine liver biopsy, including when to perform a biopsy, techniques for sample collection and handling, and strategies to optimize diagnostic yield. It discusses the interpretive framework used by pathologists, common diagnostic challenges, and prognostic scoring systems. Limitations such as sampling error and variability among observers are considered alongside ancillary testing, immunohistochemistry, and in situ hybridization. Emphasis is placed on the importance of communication between clinicians and pathologists to maximize diagnostic and prognost...
Derivatisation-free FIA-MS/MS assay for rapid simultaneous screening of atypical myopathy biomarkers and acylcarnitine profiles from dried blood spots. Atypical myopathy (AM) is a frequently fatal, toxin-induced disease in horses, characterised by acute progression and mortality rates exceeding 70% within 72 h. The disease is caused by ingestion of seedlings or seeds of certain maple species that contain the toxins hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG). Early diagnosis depends on reliable detection of these compounds and their metabolites. However, most available LC-MS/MS assays are limited by derivatisation requirements and dependence on serum and urine matrices. Here, a rapid derivatisation-free flow injection analysis...
Isolation and Identification of Emerging Equine Encephalosis Virus Serotype 6 in Israel, 2023. Equine encephalosis (EE) is an arthropod-borne viral disease resembling a mild form of African horse sickness disease, which affects all species of equids. Previously, only EEV serotype 4 (EEV-4) caused sporadic outbreaks of the disease in Israel. In the summer of 2023, EE was clinically diagnosed in horses and donkeys in Israel. For laboratory confirmation, tissue cultures were inoculated with whole-blood samples obtained from sick animals. Five EEV isolates were successfully recovered in tissue and confirmed by conventional RT-PCR and Sanger sequencing of several viral genes. BLAST analysis ...
Characterization of Capillary Zone Electrophoresis in Horses and Donkeys. There are no capillary zone electrophoresis (CZE) comparative studies between horses and donkeys. Our objectives were to compare CZE results between both species, establish reference intervals (Ris), study age- and sex-related changes, determine the prevalence of bisalbuminemia, and correlate spectrophotometric albumin results with those calculated based on two different CZE methods. We performed CZE in 50 healthy horses and 49 healthy donkeys. There were significant differences ( < 0.03) in every protein fraction percentage and concentration (except for the β-globulins percentage) between...
Theileria equi sensu lato genetic diversity and phylogeny: new insights from mitochondrion, apicoplast and nucleus multilocus gene analysis. Equine piroplasmosis is a significant tick-borne disease with a global distribution. Among the two causative agents, Theileria equi has undergone numerous taxonomic reclassifications over time, reflecting advances in our understanding of its biology, molecular characteristics, and phylogenetic relationships. Formerly named Babesia equi, now classified in the Equus group apart from Babesia and Theileria species, its genus name will again certainly change. The genetic diversity also within T. equi lead to the renaming of one lineage as Theileria haneyi. Theileria equi should better now be consid...
Activity of 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6′-methylresorufin) ester lipase in peritoneal fluid may be associated with strangulating lesions, pancreatic fibrosis, and negative outcome in horses with colic. To compare plasma and peritoneal 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR) lipase activity between horses with and without colic signs and document pancreatic histopathology in a subset of horses. Unassigned: This prospective, cross-sectional study included university-owned or donated healthy horses and client-owned horses presenting to the veterinary teaching hospital for colic. Peritoneal WBC and lactate concentrations and plasma and peritoneal DGGR lipase activity were measured. Pancreatic tissue was collected via convenience sampling. Pancreatic fibrosis ...
Venous blood lactate concentration as a predictor of intestinal viability in horses with acquired indirect inguinal hernia. Background: Acquired indirect inguinal hernia represents a life-threatening emergency. Therapeutic decision-making largely depends on intestinal viability, for which specific predictive parameters (such as venous lactate) have not been evaluated.Objectives: Investigating the predictive performance of venous blood lactate and other laboratory results on intestinal viability in horses with acquired indirect inguinal hernias.Study designRetrospective multicentre observational study.Methods: Seventy-four horses diagnosed with acquired indirect inguinal hernia (treated surgically) at two referral h...
Growth kinetics of Taylorella equigenitalis and its survival in straw-based bedding. Taylorella equigenitalis, the causative agent of contagious equine metritis, is a fastidious bacterium with limited tolerance to extra-host conditions; however, quantitative data on its growth dynamics and survival outside the host remain scarce. This study evaluated the growth of T. equigenitalis under aerobic and microaerophilic conditions in liquid medium and its survival in wet and dry straw-based bedding under controlled laboratory conditions.Growth was assessed at 20 °C and 37 °C using real-time optical density monitoring and viable cell counts. No proliferation was observed at 20 ...
Synchronization dynamics between rider and saddle motion across gaits revealed by IMU analysis. Effective rider-horse coordination is essential for performance and welfare, yet objective, field-based quantification of rider-saddle synchronization across gaits remains limited. Objective: To quantify dynamic synchronization between rider motion and saddle motion (as a proxy for horse trunk oscillations) across walk, trot, and canter using a minimal inertial measurement unit (IMU) setup, and to determine whether synchronization differs between rider body segments. Methods: Two experienced riders (8-20 years riding experience) rode their own or familiar warmblood horses (n = 2; age 6-14 year...
Establishment and evaluation of hematological and serum biochemical reference intervals in Chinese local donkey populations. Background: Reliable interpretation of hematological and serum biochemical test results in donkeys benefits from the use of population- and management-system-adapted reference intervals (RIs). However, standardized and CLSI-compliant RIs for Chinese local donkeys are still unavailable, limiting their clinical and epidemiological application. This study aimed to establish hematological and serum biochemical RIs for Chinese local donkeys using a large, multi-farm dataset, and to evaluate the effects of farm origin, age, and sex on these parameters. Blood samples were collected from healthy donke...
Conventional IVF in the horse past, present, and future: physiological and technical insights. For decades, conventional in vitro fertilization (IVF) in the horse was ineffective, leading to reliance on intracytoplasmic sperm injection (ICSI) for both research and clinical in vitro embryo production. However, the ability to achieve IVF is essential to the study of key reproductive processes, including sperm capacitation, fertilization, and early embryonic development. Recently we have established a repeatable and efficient IVF system for horses, using long-term (9-22 h) sperm pre-incubation. For clinical use, IVF has many features that may support higher embryo quality than does ICSI, a...
LC-HRMS detection of three ionizable lipids in equine plasma: surrogate markers for long-term LNP-mediated gene-doping surveillance. Lipid nanoparticles (LNPs) are essential delivery vehicles for messenger ribonucleic acid (mRNA)-based therapies. However, their potential misuse as gene-doping agents in horse racing and equestrian sports necessitates robust analytical surveillance. This study presents a highly sensitive, Orbitrap technology-driven liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for the simultaneous detection of three clinically relevant ionizable lipids: ALC-0315, SM-102, and DLin-MC3-DMA (MC3), which are critical components of approved RNA therapeutics and vaccines. Following a modi...
Comparison of equine PBMC isolation using two protocols: Impact on key cellular parameters. There are diverse uses for equine peripheral blood mononuclear cells (PBMC) in research. Studies have shown that PBMC isolation protocols can affect immune parameters of diagnostic and research assays. We aimed to assess the impact of equine blood handling techniques on cell yield, viability, and proliferation under mitogen stimulation. Whole blood (WB) samples were collected from eight healthy adult horses into heparinized tubes for PBMC isolation by comparing method 1 (M1) where freshly collected WB was layered directly over a density gradient medium for centrifugation, followed by PBMC harv...
Lacrimal caruncula thermography overestimates rectal temperature in horses: Influence of camera type and measurement distance. Rectal temperature (RT) is the gold standard for assessing body temperature in horses, but handling and welfare concerns limit its use. Thermography of the lacrimal caruncula (LC) has been proposed as a noncontact alternative, although its accuracy may be influenced by device characteristics and measurement distance. Objective: To evaluate agreement between LC thermography and RT in horses, compare two infrared thermal cameras and determine the effect of camera-to-subject distance on measurement bias and variability. Methods: LC temperature was recorded at 0.5, 1 and 2 using two cameras (IR Fl...
Integrated workflow for production and in vitro characterization of equine anti-SARS-CoV-2 hyperimmune serum with epitope mapping. This study establishes an integrated workflow linking antibody production, neutralization assessment, and epitope mapping, supporting the identification of antigenic regions recognized by polyclonal antibodies generated in vivo. The viral suspension was inactivated with β-propiolactone under biosafety level 3 (BSL-3) conditions, with no cytopathic effect or plaque formation observed, confirming loss of viral replicative capacity. The produced immunogen retained antigenic activity, as demonstrated by reactivity with IgG antibodies from vaccinated and previously infected individuals. Protein an...
Detection of papillomavirus DNA in uterine flushing samples from healthy mares reveals diverse genotypes with differing host ranges. A molecular study to assess the presence of some papillomaviruses was performed on 105 uterine flushing samples of healthy mares using real time quantitative polymerase chain reaction (qPCR) and digital PCR (dPCR). Bovine, equine, and ovine papillomavirus DNA was detected in 27 uterine flushing samples through dPCR (~ 25.71%); qPCR was able to detect 18 positive samples (~ 17.14%). Differences between the two molecular protocols were significant as shown by McNemar's test (p˂ 0.005). EcPV DNA was detected in 10 samples by dPCR (~ 9.52%) of uterine flushing samples. qPCR detected EcPV DN...
Evaluation of synthetic peptides derived from glycoproteins gp45 and gp90 as candidates for the immunodiagnosis of equine infectious anemia. The international scientific community continues to promote the development of diagnostic techniques with improved performance and novel capture antigens. On this basis, two synthetic peptides derived from gp45 and gp90 were evaluated to identify the best candidate for the indirect immunodetection of infection. In both assays, 68 serum samples from naturally infected horses in Granma Province (59 seropositive and 9 seronegative), previously classified using the agar gel immunodiffusion (AGID) test, were analyzed. The diagnostic performance of each candidate peptide was assessed by calculating ...
FGF2, LIF, and IGF1 supplementation during in vitro maturation or culture improves equine ICSI embryo development in an age-dependent manner. Ovum pickup (OPU) combined with intracytoplasmic sperm injection (ICSI) has reshaped equine assisted reproduction, enabling embryo production from mares and stallions with constrained breeding opportunities; however, limited efficiency of in vitro maturation (IVM) and in vitro culture (IVC) continues to hinder embryo production. Growth factor supplementation with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) in combination (FLI) has been shown to enhance oocyte and embryo developmental competence in several species. Objective: This...
Inter-individual variability in equine antibody responses to African snake venoms follows heavy-tailed distributions with implications for antivenom production. Variability in the antibody response of horses used for snake antivenom manufacture is well recognized, yet its statistical structure and implications for industrial productivity remain poorly characterized. In this study, we quantified antivenom antibody titers by ELISA in a cohort of 14 horses immunized with venoms from the clinically most important snakes in sub-Saharan Africa. To integrate antibody levels with plasma availability, we calculated the Cumulative Plasma Productivity (CPP) by converting individual plasma volumes into titer-corrected equivalents and sequentially pooling these vo...
Telomere-to-telomere Genome Assembly of Equus kiang (Tibetan wild donkey). The Tibetan wild donkey (Equus kiang) is an endemic equid species inhabiting the Qinghai-Tibet Plateau, exhibiting remarkable chromosomal diversity within the Equus genus. Here we present a chromosome-level, telomere-to-telomere genome assembly for E. kiang generated using Illumina short reads, PacBio HiFi long reads, and Hi-C sequencing data. The final assembly spans 4.02 Gb and comprises 27 pseudochromosomes. Specifically, 8 chromosomes are gapless, 8 chromosomes have one gap, 7 chromosomes have 2-4 gaps, and the remaining 4 chromosomes have five or more gaps. Benchmarking Universal Single...
Comparison of in vitro degradation of immunoreactive adrenocorticotropic hormone and insulin in equine blood measured by two immunoassay methods. This study compared the in vitro stability of equine plasma adrenocorticotrophic hormone (ACTH) and serum insulin measured by chemiluminescent assay (CLA; Siemens Immulite 2000XPi) and immunofluorescent assay (IFA; Tosoh AIA-900) after storage at 4°C or room temperature (approximately 21°C) for clinically relevant periods. Fresh equine EDTA blood for ACTH and coagulated blood for insulin were aliquoted and analysed immediately (T0) and after storage. ACTH was assessed at 24, 48 and 72 hours; insulin was assessed at 24 hours, 72 hours and 7 days. Percentage recovery relative to T0 was analyse...
miRNA Sequencing and Differential Analysis of Testis in 1-Year-Old and 2-Year-Old Kazakh Horses. The represents a significant genetic resource within China's population, characterized by notable resilience and an ability to thrive on coarse forage. Nevertheless, a decline in its numbers has been observed recently, making the improvement of its reproductive performance crucial for the preservation of this breed and the advancement of the related industry. In this study, testicular tissues from 1-year-old (pre-pubertal) and 2-year-old (post-pubertal) were analyzed. miRNA sequencing was conducted on tissues from these age groups, followed by bioinformatics analyses to elucidate the functi...
Allogeneic Platelet-Rich Gel Supernatant Reprograms the Cytokine and Growth Factor Microenvironment in an Equine In Vitro Inflammatory Tendon System. Tendinopathy involves a dysregulated inflammatory microenvironment in which cytokines, growth factors (GF) and extracellular matrix components interact dynamically. Platelet-rich plasma (PRP) is widely used as a regenerative therapy, but its mechanisms of action in inflamed tendon remain unclear. This study evaluated whether platelet-rich gel supernatant (PRGS) reprograms the inflammatory-anabolic mediator network in an equine in vitro tendon explant system stimulated with lipopolysaccharide (LPS). Tendon explants were cultured under six experimental conditions, including non-stimulated contro...
Validation of a stall-side immunoglobulin assay for use in equine reproductive management. Equine foals receive IgG from mare colostrum through passive transfer. Failure of passive transfer (FPT) is a significant risk to the foal's life, leaving them vulnerable to infection and sepsis. Radial Immunodiffusion (RID) and immunoturbidimetric assays quantify IgG present in a foal sample but require a laboratory to complete. Accurate, reproducible, stall-side testing to rapidly quantify IgG would allow for expedited clinical decisions, with potential to improve equine foal care and survival. Objective: To evaluate the analytical and clinical performance of a stall-side IgG lateral-flow te...
A scoping review of neuroendocrinological biomarkers with potential for use in equine behavioral medicine practice. BACKGROUND: Equine emotion and welfare are of increasing concern to the general public, equestrians and researchers. There is growing demand for more objective indicators of specific emotional states, as well as the well-being of horses in a variety of contexts. Such markers may also assist in both the diagnosis and selection of biological therapies within equine behavioral medicine. RESULTS: This scoping review summarizes and appraises the published findings relating to relevant biological measurements from the last two decades. Particular attention is given to methodological, subject-related...
