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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.
High-Throughput Equine Plasma Profiling Using Laser-Assisted Rapid Evaporative Ionisation Mass Spectrometry. Remnants of erythrocyte removal from blood constitute therapeutic plasma-a water-based solution containing mainly platelets, extracellular vesicles (EVs), protein and lipid complexes and ions. Plasma-based therapies, including their use to accelerate wound healing in human and animal patients, are increasingly gaining prominence in regenerative medicine; however, methods for the optimal preparation of individual plasma are still under development. Rapid and accurate analysis of plasma composition is essential for the development of therapeutic applications and for ensuring the quality of plasm...
Effect of lingual gauze swab placement and light exclusion on pulse oximeter agreement with arterial haemoglobin-oxygen saturation derived from blood-gas analysis in healthy anaesthetised horses. To compare peripheral arterial haemoglobin-oxygen saturation values obtained using pulse oximetry (SpO) with and without a lingual gauze swab under ambient light and light-exclusion conditions, with arterial haemoglobin-oxygen saturation derived from arterial blood-gas analysis (SaO). Methods: Prospective method-comparison study. Methods: A group of 60 healthy adult horses undergoing elective general anaesthesia. Methods: In American Society of Anesthesiologists (ASA) physical status classification I-II horses, SpO readings were obtained under six conditions during anaesthesia: no swab, no swa...
Evaluation of an Immunoturbidometric Assay for Measurement of Total Adiponectin Concentration in Equine Serum Samples. Total adiponectin (TA) is a marker of metabolic health in equids, with values below a critical decision point of < 7.9 μg/mL conferring increased risk of laminitis. Objective: To evaluate an IT assay to measure serum [TA] in equids and to assess its correlation with [high molecular weight adiponectin] (HMWA) measured by ELISA. Methods: Total serum adiponectin concentrations from 207 adult horses were measured using both an IT assay for TA and HMWA using a previously validated ELISA assay. Data were analyzed using Spearman correlation and Passing-Bablok regression. Intra- and inter-assa...
Evaluation of the Brix refractometer for diagnosing the failure of transfer of passive immunity in 12-h-old foals. Failure of transfer of passive immunity (FTPI) is the most important cause of sepsis and mortality in neonatal foals. Unassigned: This study aimed to evaluate the performance of the digital Brix refractometer for the diagnosis of FTPI in 12-h-old foals. Unassigned: Cross-sectional, diagnostic accuracy study. Unassigned: Jugular blood samples were collected from 124 foals at 12 ± 0.5 h after birth. IgG concentrations were estimated using a digital Brix refractometer and measured using the radial immunodiffusion test (RID). Correlation coefficients were calculated between serum Brix percentages...
Recombinant EHV-1 Vector Expressing Immunodominant Hemagglutinin Protein of Equine Influenza Virus H3N8 (Sub-Lineage Florida Clade 2). Equine herpesvirus type 1 (EHV-1) and equine influenza virus (EIV) are major respiratory pathogens in horses, causing significant economic losses in domesticated horses. Bacterial Artificial Chromosome (BAC) technology can be used to precisely manipulate the EHV-1 genome for the development of live-attenuated vector vaccines. Earlier, our group developed a live-attenuated EHV-1 vaccine by deleting virulence-associated genes using this technology and the mutant EHV-1 has been exploited for expressing foreign gene in the current study. Specifically, in this study, a mutant EHV-1 virus expressing...
Isolation of high-quality stallion spermatozoa: a comparison of methods and their effects on sperm quality and yield, DNA damage, and functional competence by ICSI. Intracytoplasmic sperm injection (ICSI) is the primary method for equine in vitro embryo production, yet outcomes may be influenced by sperm selection techniques. Objective: This study compared four isolation methods: EquiPure™, Vetmotl™, Felix™, and Samson™, to assess effects on sperm quality and embryo development. Methods: Semen was collected from four fertile stallions, processed and evaluated for sperm motility, viability, DNA integrity, and yield of high-quality cells relative to input. Isolated samples were compared to non-isolated (centrifuged) samples. Spermatozoa were used fo...
Assessment of Thyroid Hormones Using the Immulite 2000xpi Analyzer in Healthy Donkeys. The Immulite 2000xpi, a chemiluminescent immunoassay (CLIA)-based analyzer, is currently recommended for measuring thyroid hormones (TH) concentrations in equids. However, scarce data are available on TH measurements in donkeys using this technique. Similarly, the effects of factors such as age and gender on TH concentrations have not been assessed with this analyzer. Serum free and total T4 and T3 concentrations were measured in 40 healthy donkeys (6.1 ± 0.8 years old; 8 intact jacks and 32 jennies) and 41 horses (10.7 ± 0.5 years old; 15 stallions and 26 geldings) using the Immulite 2000xp...
Reference interval for serum amyloid a in apparently healthy donkeys measured with a donkey-specific ELISA. Serum amyloid A (SAA) is a major acute phase protein used as an inflammatory biomarker in equine medicine. However, no universally accepted assay-specific reference interval (RI) exists for donkeys, and available literature remains limited, with only few studies using different donkey breeds and immunoassay platforms. This study aimed to establish an assay- and population-specific RI for serum SAA in apparently healthy donkeys using a species-specific double sandwich ELISA. Unassigned: Serum SAA concentrations were measured in 176 apparently healthy donkeys. RI estimation followed ASVCP/CLSI r...
Simple and efficient HPLC-MS/MS method for simultaneous extraction and quantification of anthelmintics in equine manure. Anthelmintic treatment is a common practice in horses. The presence of pharmaceutical residues in fields and pastures is well-documented affecting various organisms, like nematodes. This study describes the development and validation of a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method using multiple reaction monitoring (MRM) for extracting and determining the concentrations of four commonly used anthelmintics in horse manure. Manure was initially dried, then spiked with various concentrations (10-10,000 ng) of anthelmintics and extracted using an acetonit...
Identification of a Novel Slow-Gray STX17 Lineage in Japanese Thoroughbreds via a Multi-tiered Copy Number Analysis Workflow. Gray is a dominant coat color phenotype in horses caused by a ~ 4.6 kb tandem triplication within intron 6 of syntaxin 17 (STX17). The copy number variation (CNV) of the duplicated segment influences the graying rate. The rare G2 allele (CNV = 2) is associated with a slower graying rate compared to the common G3 allele (CNV = 3) and is also relevant to melanoma risk. Current assays are limited because long and accurate PCR (LA-PCR) detects only the presence or absence of duplications, while droplet digital PCR (ddPCR) cannot reliably distinguish certain genotypes such as G3/g and ...
Sperm Energy-restriction and Recovery (SER) enhances equine sperm function and embryo development after ICSI. Assisted reproductive technologies (ART) are extensively used in domestic animal reproduction; however, improving their efficiency and consistency remains a major challenge for achieving reliable reproductive outcomes. The equine industry faces unique challenges related to sperm capacitation, which limited the success of conventional in vitro fertilization (IVF) for many years. Consequently, intracytoplasmic sperm injection (ICSI) became the most widely used technique for commercial in vitro embryo production. Even though a successful IVF protocol was recently developed, substantial opportunit...
Development of lateral flow assays for the rapid diagnosis of glanders in equids. Glanders is a contagious zoonotic disease caused by that primarily affects horses, donkeys, and mules. It remains endemic in parts of Asia, Africa, the Middle East, and South America, where increasing case reports highlight the need for accessible diagnostic tools. Conventional diagnostic methods are limited by high costs and specialized laboratory requirements, whereas lateral flow assays (LFAs) offer practical, user-friendly, and rapid alternatives. In this study, we developed three LFAs targeting antibodies against the antigens Hcp1, GroEL, and whole-cell lysate (WCL), previously identifi...
Influence of collection site on cerebrospinal fluid test results in horses with equine protozoal myeloencephalitis. Cerebrospinal fluid (CSF) analysis is a critical component in the diagnosis of equine protozoal myeloencephalitis (EPM). Test results obtained may differ based on different collection sites. Objective: Compare results of EPM-specific diagnostic tests obtained from different collections sites in EPM-affected and non-EPM affected horses. Methods: Twenty control and 7 EPM-affected horses. Methods: Prospective observational study. CSF was collected from the lumbosacral (LS) and cranial sites (atlantooccipital or C1-2) in 27 horses and EPM-specific diagnostic tests were performed. Data were summari...
Factors Influencing Equine Embryo Outcomes in OPU‑ICSI Systems: Insights from a Large‑Scale Program in Brazil. Equine OPU‑ICSI supports commercial IVEP, yet substantial variability persists. We retrospectively analyzed a large dataset generated in Brazil spanning Quarter Horses, Warmbloods, and Mangalarga to evaluate whether predictors previously identified in the literature show comparable patterns under distinct ecological and breeding conditions. Overall oocyte and embryo outcomes were aligned with broadly reported trends from previous studies. Oocytes retrieved from estrous mares bearing a dominant follicle exhibited higher maturation and blastocyst rates. Stallion effects segregated into distinc...
Antibiofilm action of ozonated sunflower oil against bacteria isolated from the uterus of mares susceptible to endometritis. This study aimed to characterize the antibiofilm action of ozone-enriched sunflower oil (O) against pathogens associated with endometritis in mares. Initially, samples from 41 mares with a history of subfertility were stored in BHI broth and 0.9% NaCl solution. The samples underwent microbial culture, identification, and sensitivity testing at LABRAPE-UFRPE, Brazil. Biofilm production was evaluated by the crystal violet method. Subsequently, biofilm-forming bacteria were subjected to the action of different concentrations of ozonated sunflower oil. Of the 21 isolates tested for biofilm produc...
Rapid bacterial community profiling of equine faecal, skin, milk and saliva samples using Oxford Nanopore long-read 16S rRNA amplicon sequencing. The composition of the equine gut microbiome is associated with many aspects of gastrointestinal, respiratory and musculoskeletal health that have been reported in the horse. Scientific studies exploring the microbiome non-intestinal ecological niches in or on horses are lacking. The clinical use of bacterial community profiling in horses is currently limited by cost and by slow analytical workflows. Most equine microbiome studies have relied on 16S rRNA amplicon sequencing of bacterial DNA, using high-throughput short-read sequencing technologies. This is often provided by an external servic...
Thoroughbred horses susceptible to Recurrent Exertional Rhabdomyolysis have elevated skeletal muscle mitochondrial capacities. Recurrent Exertional Rhabdomyolysis (RER) is a skeletal muscle disease that can intermittently impede careers of equine athletes. Manifesting as episodes of muscular stiffness and degeneration, RER causes lost training and competition days. RER is associated with abnormal intracellular calcium (Ca) regulation. Calcium released from the sarcoplasmic reticulum to initiate contraction may also be transported into mitochondria, which impacts energy production. RER-susceptible horses exhibit down-regulated mitochondrial proteins but upregulated mitochondrial genes. Thus, mitochondrial function may ...
Prevalence of insulin dysregulation in a sport horse population as determined by traditional and stall-side testing. The prevalence of insulin dysregulation (ID) has not been investigated in sport horses. Stall-side insulin assays offer convenience but need further validation. Objective: To determine the prevalence of and risk factors for ID in a sport horse population using an oral sugar test (OST) and assess agreement between stall-side and validated laboratory assays for insulin quantification. Methods: Prospective cross-sectional study. Methods: One hundred eighty sport horses presenting for orthopaedic evaluation at a university hospital (N = 140) or local farms (N = 40) were tested for ID using...
Diagnosis of bacteraemia in neonatal foals using 16S rRNA high-throughput sequencing. Sepsis is an important cause of morbidity and mortality in foals. Early diagnosis can improve outcome but is complicated by non-specific clinical signs and delayed confirmation of infection via blood culture. Molecular assays represent a rapid and more sensitive alternative. Objective: To evaluate and compare bacterial load and composition in blood and blood culture media (BCM) of sick and healthy foals using molecular assays and to compare results with traditional bacterial culture. Methods: Cross-sectional observational clinical study. Methods: Thirteen septic foals, 10 sick non-septic and 8...
Circulating Elements in the Bloodstream of Growing Foals: Focus on Heavy Metals and Bone Remodeling Across Growth Stages. Bone tissue undergoes continuous remodelling throughout the life of the individual, and growth is the most impacting phase. In fact, tightly coordinated processes of resorption, synthesis, and mineralization of the bone matrix under systemic hormonal, biomechanical, and metabolic control occur for variable periods of time across species. The horse is a fast-growing animal and for this reason identified as precocial. It was hypothesized that blood serum levels of minerals in growing foals at different stages of accretion may vary over time. This investigation aimed to assess the dynamics of cir...
Matched embryo-endometrium RNA-seq reveals coordinated but asymmetric transcriptomic reprogramming at the onset of early equine pregnancy. Maternal recognition of pregnancy (MRP) in the mare requires coordinated adaptations of both the conceptus and the endometrium during the peri-recognition interval; however, integrated transcriptomic analyses of both compartments within the same biological window remain limited. Here, we analyzed conceptus RNA-seq data across embryonic age (Days 8 and 12 post ovulation) and endometrial RNA-seq data across pregnancy status (pregnant versus non-pregnant mares sampled between Days 8 and 12 post ovulation). In pregnant mares, conceptus recovery and endometrial biopsy collection were performed with...
Forage nutritive analysis in practical equine feeding: challenges and opportunities. Previous research has shown that forage nutritive analyses are not used to any large extent in equine nutrition. As forages differ widely in nutritional quality, knowledge of the nutritive value is essential to avoid under- or overfeeding of horses. Objective: The aim of this study was to investigate how forage nutritive analyses were used among horse owners in Sweden, and to identify challenges horse owners encounter with such analyses. Methods: Data was collected through an online survey with questions about the respondent, access to forage analytical reports, if and how the respondent used ...
Development of a rapid and highly accurate diagnostic test for detecting Theileria equi in horses using recombinase polymerase amplification (RPA) combined with nucleic acid lateral flow immunoassay. Equine piroplasmosis (EP) is a protozoan disease in horses, mainly caused by Theileria equi, and is associated with non-specific signs such as fever, anemia, and edema. The lack of specific clinical signs, coupled with the high prevalence of carrier horses, highlights the critical need for a rapid and highly accurate diagnostic test for the detection of Theileria equi. Existing methods, including molecular, serological, and microscopic techniques, require specialized equipment, skilled personnel, and considerable time. The current study aims to develop a rapid and accurate assay for the detect...
Feasibility and safety of repeated 23% hypertonic saline boluses for electrical impedance tomography contrast enhancement in anaesthetised horses. Hypertonic saline (HS) has been used to enhance electrical impedance tomography (EIT) cardiac-related signals associated with pulmonary perfusion in other species, but its suitability and physiological effects in horses are unknown. This exploratory study aimed to determine whether intravenous administration of 100 mL of 23% HS produces a detectable change in thoracic impedance in anaesthetised horses, and to evaluate the physiological effects of repeated bolus administration. Six adult horses were anaesthetised twice using total intravenous anaesthesia. Hypertonic saline 23% was administered ...
Exploration of the relationship between laboratory stress parameters and leukocyte coping capacity in ponies exposed to repetitive handling during a research project. Stress is an umbrella term indicating increased allostatic load with the potential to reach a pathological state of the organism. In this context, cortisol concentration and neutrophil granulocytes-to-lymphocyte ratio (NEU: LYM) are expected to increase. On the functional pathway, stress reduces the oxidative burst capacity of neutrophils and therefore, the leucocyte coping capacity (LCC) quantifying this oxygen radical generation of neutrophils. Therefore, we hypothesized that in ponies subjected to repetitive handling, LCC will decrease and will be negatively correlated with cortisol concent...
Towards a Comprehensive Screening Approach for Detecting Gene Doping in Racehorses and Sport Horses by Targeting Common and Intrinsic Plasmid Sequences. The evolution of biotechnology and gene therapy has unfortunately facilitated the emergence of gene doping. Those misused applications are prohibited in equestrian sports (International Equestrian Federation (FEI)) and in horseracing (International Federation of Horseracing Authorities [IFHA]), reflecting major concerns regarding horse welfare and the integrity of competitions. Among the biotechnological tools that could be misused, eukaryotic expression plasmids represent a particular threat due to their ease of production, customisable design and ability to carry a wide range of performance ...
Evaluation of oxidative stress in ill neonatal foals: a preliminary study. Oxidative stress both contributes to and results from sepsis pathophysiology, but its role in ill foals remains poorly characterized. Objective: Ill foals will exhibit decreased antioxidant defense activity and increased oxidative products compared with healthy foals, and these changes will be associated with higher sepsis scores, lower survival scores, and non-survival. Methods: Twenty-six foals < 14 days old: 16 hospitalized foals (11 sick non-septic, 5 septic; median age, 1.5 days; range 1-13) and 10 healthy Standardbred foals (2 days old). Nine of 16 ill foals survived to discharge...
Effect of Spirulina platensis extract (C-phycocyanin) supplementation to the semen extender on kinematic and oxidative parameters of Marwari stallions during freezing. The current research aimed at examining the impact of different quantities of Spirulina platensis extract (C-phycocyanin) supplemented to the semen extender on the post-thaw quality of Marwari stallion spermatozoa. Semen samples were obtained via an artificial vagina from six fertile stallions and diluted with an extender containing Spirulina platensis extract (C-phycocyanin) at concentrations of 10, 20, and 50 µg/mL, or without Spirulina as a control. Aliquots of diluted semen were subjected to cooling (4 °C, 120 min) and then frozen (-196 °C, 1 day), after which the kinematic paramet...
Cracks in the ice: biological challenges and incremental progress in equine oocyte vitrification. Equine oocyte vitrification could enhance the flexibility of in vitro embryo production, yet developmental competence remains reduced. Only vitrification of oocytes matured in vivo yields foaling rates comparable to fresh oocytes. Recent advances in vitrification at the germinal vesicle (GV) stage, supported by systematic comparisons of cryoprotective agents and exposure times, has enabled consistent blastocyst production and birth of several foals. Comparative studies of GV and metaphase II (MII) stages suggest biological advantages of vitrification after in vitro maturation (IVM), warranting...
Evaluation of Point-of-Care Coagulation Measurement Systems for Activated Clotting Time and Partial Thromboplastin Time in Heparinized and Non-Heparinized Equine Blood. To compare the performance of one point-of-care activated clotting time (ACT) machine (Vet-Tube), one point-of-care activated partial thromboplastin time (aPTT) machine (Coagulation DX), and one bench-top ACT machine (Medtronic ACT II plus) with previously published measurements from stationary devices in whole blood from healthy horses. Methods: Prospective, observational study SETTING: University veterinary teaching hospital, from August 2023 to September 2023. Methods: Thirty healthy university-owned horses. Methods: ACT and aPTT were measured via point-of-care analyzers (ACT POC, aPTT POC)...
