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.
Evidence That GYPA (Glycophorin A) Encodes the K Blood Group System in Horses. Although serological and genetic studies of equine blood group systems have been conducted for many years, the molecular basis of erythrocyte antigens' variability has remained largely unexplored. In this study, we aimed to elucidate the genetic basis of serological variation within equine blood group K. Using mRNA extracted from peripheral blood samples (n = 100) collected from horses with known serological blood types (Ka or K-), we performed a transcriptome-wide association study (TWAS), which revealed a significantly associated region on equine chromosome 2 (ECA2). A detailed analysis ...
Comparative Evaluation of Sperm Quality Assessment Methods in Purebred Spanish Horses. Evaluating semen quality in Purebred Spanish Horses is essential to determine reproductive potential and optimize assisted reproductive techniques. Objective: This study compared several sperm analysis methods in Purebred Spanish Horses to standardize protocols suitable for both laboratory and field conditions. Methods: Fifteen stallions were evaluated through three ejaculates, analyzing sperm parameters using different techniques. Sperm kinematics was assessed by Sperm Computer Analyzer (SCA-CASA) or iSperm mCASA. Viability was analyzed by eosin-nigrosin-Giemsa (ENG) staining or Hoechst 33258...
Effect of Acer species (red and Freeman maple) and horse characteristics, management, and location on the in vitro oxidation of equine erythrocytes. Red maple toxicosis arises when horses consume wilted maple leaves, resulting in methemoglobinemia and hemolytic anemia due to oxidative damage to erythrocytes. Objective: The objective of this study was to determine how maple species (red versus Freeman maple) and horse characteristics, management, and barn site contributed to in vitro methemoglobin production and hemolysis. Methods: Horse information was obtained through an owner questionnaire. Extracts from red and Freeman maple leaves were separately incubated with washed erythrocytes from each horse (n=120). The in vitro percentage methem...
Supramolecular Solvent Extraction for Doping Control Analysis of Prohibited Substances in Horse Urine. Despite the recent success in introducing supramolecular solvents (SUPRAS)-based extraction to drug analysis, its application and robustness in day-to-day regular urine testing have yet to be demonstrated. Moreover, the applicability of SUPRAS in equine doping control testing remains unexplored. In this work, we have successfully developed for the first time a simple, rapid, inexpensive, and environmentally friendly SUPRAS extraction method for analyzing 76 prohibited substances of different classes (selective androgen receptor modulators, hypoxia-inducible factor prolyl hydroxylase inhibitors...
Characterization of isolated late preantral and early antral equine ovarian follicles. Understanding how to effectively isolate a large number of healthy follicles is essential for increasing success rates in ARTs, such as in vitro culture and cryopreservation. Late secondary (preantral) and early tertiary (antral) follicles isolated from the intermediary portion of equine ovarian tissue sliced at two different thicknesses (0.25 mm vs. 0.50 mm) were characterized according to regional distribution (dorsal, medial, and ventral), morphofunctional features, and epigenetic profiles. After isolation via microdissection, follicles were evaluated for membrane integrity (trypan blue), v...
Reference intervals for serum macro- and microminerals in clinically healthy horses in Northwestern Spain: Influence of age, sex, breed and diet. Serum mineral concentrations are key indicators of nutritional status, metabolic function and health in horses; however, contemporary reference intervals (RI) derived from large, well-characterised populations remain scarce. Objective: To establish serum RI for macro- and microminerals in clinically healthy horses in Northwestern Spain and to evaluate the influence of sex, age, breed and diet on mineral status. Methods: Blood samples were collected from clinically healthy horses (n = 211), and serum concentrations of essential macrominerals (calcium [Ca]; phosphorous [P]; magnesium [Mg]; sodiu...
Re-emergence of equine glanders in Mongolia based on region-wide serosurveillance. Glanders, a zoonotic disease caused by , has re-emerged in Mongolia after decades of control. We conducted nationwide serosurveillance from 2021 to 2024 to assess the prevalence and geographic distribution of glanders. Using random 3-stage cluster sampling, we collected 3,001 horse serum samples from 332 herds. Those herds were in 46 soums (administrative districts equivalent to counties) in 13 provinces and in 3 districts in Ulaanbaatar. Sera were tested using the complement fixation test, and positive results were confirmed by immunoblotting and ELISA. Overall seroprevalence was 1.03% (95% C...
Evaluation of oxidative stress and antioxidant defense biomarkers in healthy and colic horses: correlation with type of colic and outcome. Colic is a major cause of morbidity and mortality in horses, with oxidative stress implicated in its pathophysiology. Objective: Evaluate biomarkers (BIOs) of oxidative stress and antioxidant defense in healthy horses and those with non-strangulating colic (NSC) and strangulating colic (SC) and assess correlations with survival. Methods: Seventy-one adult horses: 10 healthy and 61 colic-affected (42 NSC, 19 SC) admitted to 3 veterinary teaching hospitals. Methods: Prospective, multicenter cohort study. Blood samples were collected at admission (T0) and up to 96 h post-admission. Biomarkers me...
Isolation and characterization of seminal extracellular vesicles subsets and their impact on sperm freezability in stallions. The ability of spermatozoa to withstand cryopreservation differs between stallions. While the underlying mechanisms of these differences are not fully understood, seminal plasma (SP) may play a crucial role in modulating sperm cryotolerance. Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs), nanometer-sized membrane particles that can transfer biomolecules to sperm modulating their function. This study aimed to isolate and characterize two sized-sEV subsets-small (S-) and large (L-)-from stallion SP, and to evaluate their involvement in sperm freezability. Sem...
Serial postoperative peritoneal fluid analyses in horses with naturally-occurring strangulating and non-strangulating gastrointestinal lesions. To describe characteristics of postoperative peritoneal fluid following exploratory laparotomy for naturally-occurring gastrointestinal lesions in horses. Methods: Prospective, observational cohort study. Methods: A total of 26 client-owned horses that underwent exploratory laparotomy for naturally-occurring gastrointestinal lesions. Methods: Abdominocentesis was performed pre- or intraoperatively, and at three time points postoperatively (24, 72, and 168 h). Peritoneal lactate, total protein (TP), total nucleated cell count (TNCC), cytology, and systemic lactate were performed at each time ...
Metabolomics reveals early predictors of blastocyst formation in equine ICSI-derived embryos. Equine in vitro embryo production (IVP) via ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) has gained prominence in sport-horse breeding due to high success rates, efficient semen utilization, and year-round embryo production, including from mares with fertility challenges or postmortem. However, assessing viability of early-stage ICSI-produced equine embryos remains challenging due to subjective morphological evaluations and limited visibility of developmental features. Non-invasive metabolomic analysis of culture media offers a promising alternative to enhance embryo selectio...
Optimising Camera-ChArUco Geometry for Motion Compensation in Standing Equine CT: A CT-Motivated Benchtop Study. Standing equine computed tomography (CT) acquisitions are susceptible to residual postural sway, which can introduce view-inconsistent motion and degrade image quality. External optical tracking based on ChArUco fiducials is a promising, low-cost strategy to enable projection-wise motion compensation, yet quantitative guidance on how camera-marker geometry affects pose-estimation performance remains limited. This CT-motivated benchtop study characterizes how the relative camera-ChArUco configuration influences both the accuracy (bias with respect to ground truth) and the precision (repeatabili...
In vitro anthelmintic activity of extracts and bioactive compounds from Azadirachta indica and Cnidoscolus angustidens on strongyles L3 larvae in horses. Gastrointestinal endoparasitic diseases, particularly those caused by some strongyles, represent one of the main health challenges in equine production. The prolonged use of synthetic anthelmintics has led to the development of resistance, which has prompted the search for more sustainable therapeutic alternatives. However, plant extracts rich in secondary metabolites are emerging as a promising option. Objective: The objective of this study was to evaluate the in vitro anthelmintic activity of aqueous extracts and bioactive compounds (total phenols and saponins) from Azadirachta indica and Cn...
Beyond the host: Unveiling the independent microbiome of equine gastrointestinal nematodes. Gastrointestinal nematode infections significantly impact equine health and welfare, with rising anthelmintic resistance demanding alternative control strategies. Emerging evidence suggests that parasitic nematodes harbour distinct microbiomes, potentially influencing host-parasite dynamics and parasite survival. This study aimed to characterize and compare the microbiomes of equine gastrointestinal nematodes and their hosts, focusing on differences in composition, diversity, and core microbiota structure across different intestinal sites, nematode subfamilies, and sexes. Faecal and nematode s...
Enhancing African horse sickness virus detection: comparing and adapting PCR assays. African horse sickness (AHS) is the only equine disease for which the World Organisation for Animal Health (WOAH) gives official disease-free status, given that it poses a major threat to the equine industry. The disease is caused by AHS virus (AHSV; family , taxon species ), which is endemic in sub-Saharan Africa. Reverse-transcription quantitative real-time PCR (RT-qPCR) is a rapid, sensitive detection method used in the diagnosis of AHS and the certification of animals as negative for AHSV for the purpose of movement. Genetic variability of AHSV may influence the accuracy of RT-qPCR detecti...
Nicotinic acid treatment improves the developmental potential of equine oocytes for cloned embryo production. Nicotinic acid (NA) treatment during in vitro maturation (IVM) has been shown to elevate nicotinamide adenine dinucleotide (NAD) levels and improve oocyte developmental competence. Suboptimal equine oocyte IVM systems currently limit the efficiency of viable embryo in vitro production. This study evaluated NA supplementation during IVM for cloned equine embryo production, using oocytes from abattoir-sourced ovaries and live mares via ovum pick-up (OPU). Abattoir-derived oocytes (n = 694) were treated without or with 50 or 200 μM NA during the 18 h holding period (Pre-IVM). Next, OPU-deriv...
Prognostic Significance of Erythrocyte Sedimentation Rate for Survival in Equine Colic. Accurate early prognostic assessment is a central aspect of clinical decision-making in horses presenting with colic. Despite the availability of multiple clinical and laboratory parameters, reliable biomarkers that provide useful information at the time of admission remain limited. The erythrocyte sedimentation rate is a non-specific indicator of systemic inflammation that has recently been validated for automated point-of-care use in horses. Its behaviour in equine colic has not been previously characterised. This study included 85 horses admitted for colic at two university referral hospita...
Generation and characterization of an immortalized equine osteoblast cell line. Bone is a dynamic tissue that is maintained through the co-ordination of bone resorption and bone formation. An imbalance of these processes can lead to bone disease. In vitro studies of osteoblasts can help to understand bone formation, but primary cells have a limited lifespan in culture. Herein, we report the successful generation of equine immortalized osteoblasts through the stable overexpression of human telomerase reverse transcriptase (hTERT) and Simian virus 40 (SV40) large T-antigen in osteoblasts isolated from trabecular bone taken from the third metacarpal of a two-year-old Thoroug...
A Novel Equine F[ab’]2 Veterinary Antivenom for North American Viperid Snake Envenomation Demonstrates Efficacy by Rapid Serum Venom Removal and Improvement in Snakebite Severity Score. To assess the efficacy of a new equine F[ab'] antivenom (US Department of Agriculture code 6101.05) in removing serum venom and lowering the snakebite severity score (SSS) in animals suffering from naturally occurring viperid envenomation. Methods: Retrospective study, April 2020 to October 2021. Methods: Veterinary hospitals across four North American regions. Methods: Twenty-two dogs, one cat, and one horse. Methods: All patients received the new antivenom intravenously and were assessed for clinical response. Most dogs required one vial for clinical control of signs. Results: Among the dogs...
Evaluation of oxidative stress in the newborn foal: a multibiomarker approach. During the perinatal period, neonates experience a sudden increase in oxygen exposure, predisposing them to oxidative stress. Objective: This prospective, in vivo study aimed to evaluate biomarkers of oxidative stress and antioxidant defence in healthy neonatal foals during the first 48 hours of life. Methods: Ten Standardbred foals were enrolled. Blood samples were collected at birth, 24 hours, and 48 hours after birth and analyzed for biomarkers using spectrophotometry assays. Statistical analysis was performed using non-parametric tests and linear regression. Results: The results revealed s...
Comparative Analysis of Extracellular Vesicle Isolation From Equine Serum and Plasma Using Two Isolation Methods With Structural and Proteomic Validation. Extracellular vesicles (EVs) are promising biomarkers and mediators of intercellular communication, but their isolation from equine biofluids remains challenging. This study compared two isolation workflows-size-exclusion chromatography (SEC) and differential ultracentrifugation followed by SEC (UC + SEC)-to evaluate their efficiency, reproducibility, and the proteomic composition of EVs derived from equine serum and plasma. Blood from six healthy horses was processed to obtain platelet-free plasma and serum. EVs were isolated using SEC or UC + SEC and characterized by transmission ele...
Detection and Longitudinal Monitoring of Cyclosporine in Horse Plasma and Urine After Ocular Implant Administration. This paper describes the detection and longitudinal monitoring of cyclosporine in plasma and urine after subconjunctival implant administration in a horse. Sensitive liquid chromatography tandem mass spectroscopy (LC-MS/MS) methods for detecting cyclosporine in horse plasma and urine have been developed and validated, with estimated limits of detection down to 1 pg/mL in both matrices. The developed methods enabled longitudinal monitoring of cyclosporine levels in blood and urine samples collected over 6 months from a horse that had received an ocular cyclosporine implant.
Sequencing and Analysis of mtDNA Genomes from the Teeth of Early Medieval Horses in Poland. This study presents the sequencing and analysis of mitochondrial DNA (mtDNA) genomes from nine early medieval horse remains excavated across archaeological sites in Silesia region in present day Poland. Methods: Using aDNA extraction protocols optimized for short fragments, combined with target enrichment and high-throughput sequencing, we reconstructed partial mtDNA sequences for seven of the specimens. Results: The authenticity of the aDNA was confirmed through damage pattern analysis. Phylogenetic reconstruction revealed that the specimens belonged to six distinct mtDNA lineages (B, D, E, G...
Effects of sample storage time and temperature on equine complete blood counts. Equine blood samples are often collected in uncontrolled, distant environments and stored for later analysis. Objective: This study aimed to determine how sample storage methods influence complete blood count (CBC) parameters, hypothesizing that temperature and time elapsed from collection to analysis would affect CBC parameters. Methods: Blood samples were collected from 10 healthy Quarter Horse mares (age: 13 ± 4.5 years old) and stored in EDTA tubes at room temperature (22°C), above ice (8°C), refrigerated (4°C), or in contact with ice (2°C). The IDEXX ProcyteDx was used to obtain CBCs...
Functional and molecular characterization of equine intestinal organoids across media conditions and intestinal segments. Gastrointestinal (GI) disease is a major cause of morbidity and mortality in horses, with disruption of the intestinal epithelial barrier playing a central role in disease pathogenesis. A deeper understanding of the molecular and functional properties of the equine intestinal barrier is essential to improve diagnostics and therapeutics. While intestinal organoids have emerged as a promising tool for modeling GI physiology and disease, equine-specific data remain limited. Existing studies vary in methodology and often lack functional characterization, particularly across different intestinal re...
Cell Surface Vimentin Is an Attachment Factor That Facilitates Equine Arteritis Virus Infection In Vitro. Our laboratory identified the susceptible allelic variant of equine CXCL16 protein (EqCXCL16S) as an entry receptor for equine arteritis virus (EAV). However, EAV has a broad host cell tropism and infects cells that lack EqCXCL16S. Thus, we hypothesized that EAV interacts with other host cell protein(s) that facilitate EAV infection. A virus overlay protein-binding assay in combination with a Far-Western blot from EAV-susceptible equine pulmonary artery endothelial cells (EECs) and equine dermal fibroblasts (E. Derm) identified a 57 kDa protein, present in the membrane fraction of the protein ...
Serological Assays to Measure Rabies Antibody Response in Equine Serum Samples. Rabies is a neglected tropical zoonotic disease caused by rabies-virus (RV) infection and is responsible for almost 60,000 annual deaths globally, largely affecting the socio-economically disadvantaged population. Although fatality is preventable by immunization either before or after exposure with therapeutic antibodies, the high cost of prophylaxis or treatment limits their accessibility for the affected population. However, due to the almost 100% fatality rate in symptomatic individuals, almost 29 million annual vaccinations are performed, imposing high financial burden. Human transmission ...
Clinical and laboratory evaluation in horses submitted to intracecal fluid therapy administered in two different rates. Two routes of administration for electrolyte solutions are commonly used in horses with fluid, electrolyte, and acid-base imbalances: intravenous and nasogastric. Despite the frequent use of these routes, there are situations in which they cannot be applied. In such cases, intracecal fluid therapy represents a viable alternative, as this route enables the administration of enteral electrolyte solutions even in animals lacking normal anterior gastrointestinal function. This study aimed to evaluate the effects of a neutral hypotonic enteral electrolyte solution administered intracecally at two d...
Evaluation of Laboratory Techniques for the Diagnosis of Leptospira-Associated Equine Recurrent Uveitis (ERU) With Focus on the Goldmann-Witmer Coefficient. To evaluate different laboratory procedures for determining the etiologic diagnosis of equine recurrent uveitis regarding intraocular infection with Leptospira spp. and to establish a diagnostic guideline. Methods: Eighty horses with a history of ERU were ophthalmologically examined. Serum and aqueous humor were collected. Total protein, albumin level, and MAT against Leptospira spp. were evaluated on serum and aqueous humor. PCR for Leptospira spp., EHV-1 and -4 was performed on aqueous humor. Goldmann-Witmer coefficient (GWC) and C-value (CC) were calculated based on MAT. In 42 cases, an add...
Investigation of gene stability in equine luteal tissue during mid-diestrus phase and early pregnancy – Research Article. Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a technique that allows for the semi-quantification of mRNA transcripts present within a tissue of interest. Differences in the relative abundance of mRNA between samples detected by RT-qPCR require normalization with a reference gene or genes whose transcript abundance is stable within the tissue of interest independent of experimental conditions. In the field of equine reproductive studies, ACTB, GAPDH and B2M genes are the most widely used as reference genes for the normalization of RT-qPCR results. However,...