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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.
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...
Efficacy of an iron absorption test on assessing iron status in horses. Iron absorption tests (IATs) are used to evaluate iron absorption in humans. However, this testing has not been conducted in horses. Objective: The objective was to assess how a large dose of iron affects blood iron parameters during an IAT. Methods: Mixed-breed geldings (n=12) were offered 2% of their bodyweight (BW) in mixed-grass hay and an iron-free vitamin-mineral supplement. A baseline IAT (Baseline IAT) was performed, where horses received 25 milligrams per kilogram (mg/kg) of BW of iron (ferrous sulfate) orally. Jugular venous samples were taken before the iron dose, and 3-, 6-, 12-, a...
Transference of diagnostic thresholds for equine plasma ACTH from the Immulite 1000 to Immulite 2000XPi and Tosoh AIA systems. Diagnosis of PPID in horses relies on measurement of plasma adrenocorticotrophic hormone (ACTH) concentrations. Because measured ACTH values vary between analytical platforms, and published diagnostic thresholds apply almost exclusively to the Immulite 1000 chemiluminescent assay (CLA), there is a need for validated, assay-specific thresholds for contemporary analysers. This study aimed to transfer and validate established Immulite 1000 diagnostic thresholds for plasma ACTH to three contemporary systems (Immulite 2000XPi, Tosoh AIA-900, and Tosoh AIA-360) using the Clinical Laboratory Standard...
ProAKAP4 as a predictor of semen freezability in Asturcon ponies. Cryopreservation is a valuable tool in equine reproduction, although its success remains highly variable among stallions. The proAKAP4 protein has been proposed as a molecular marker of sperm quality and cryotolerance. Objective: This study aimed to evaluate the relationship between proAKAP4 concentration and sperm cryotolerance in ejaculates from Asturcon ponies, and to assess whether proAKAP4 levels in fresh semen can predict post-thaw sperm quality parameters before and after cryopreservation. Methods: Twenty-four ejaculates from eight different stallions were used (three ejaculates per ani...
Equine Oocyte and Embryo Metabolism in the ART Setting: Current Knowledge and Clinical Significance. Equine in vitro embryo production has become very efficient and is used extensively worldwide for the clinical production of foals. What we know so far about offspring health and performance is promising; however, elements of the in vitro process remain unphysiological when compared to in vivo conditions. Studying the metabolism of oocytes and embryos can help inform the optimisation of culture systems and identify aberrations if they occur. Given the growing body of knowledge on the downstream effects of an inadequate peri-conception environment, both in vivo and in vitro, it is imperative th...
Holding immature equine oocytes for up to 3 nights prior to ICSI under practical shipping conditions (10-22 °C) is compatible with blastocyst development. In vitro embryo production via intracytoplasmic sperm injection (ICSI) is widely used in equine reproductive management. Shipping of immature oocytes allows oocyte recovery for ICSI to be performed by referring veterinarians. However, delays in oocyte shipment are common, and their effect on outcomes is unclear. Shipping is typically done at about 22 °C, but previous studies have shown that holding at 15 °C maintains equine oocyte developmental competence. We hypothesized that lower temperatures may better support oocyte viability if the case that delays occur. Oocytes (n = 664) were rec...
Genomic diversity and structure in arabian horses revealed by whole-genome sequencing: establishment of an allele frequency database of common genetic variation. BACKGROUND: The Arabian horse is a culturally and historically influential breed that has contributed to the development of many modern horse populations. However, genomic resources for this breed remain limited, particularly population-level allele frequency datasets that support studies of genetic diversity, selection, and disease. The aim of this study was to generate a comprehensive allele frequency database for Arabian horses using representative sampling and high-coverage whole-genome sequencing. RESULTS: We generated the first publicly available allele frequency database for the Arabian...
Presurgical acetone and chlorhexidine aseptic preparation of the equine hoof lowers bacterial counts below surgically acceptable threshold. The objectives were to determine whether topical applications of acetone and 4% chlorhexidine following removal of the superficial hoof wall reduces tissue bacterial load below 5 log10 microorganisms per gram (CFUs/g) of tissue and whether Dey-Engley neutralizing broth (DENB) neutralizes residual chlorhexidine in vitro within equine hoof tissue samples. Unassigned: In a clinical prospective study with a total of 24 hooves, the superficial hoof was mechanically removed followed by disinfection with acetone and 5-minute rough and aseptic preparations with 4% chlorhexidine. Dorsal wall, frog, and...
Evaluating Protein Enrichment Methods to Improve Biomarker Discovery in Equine Cerebrospinal Fluid. Cerebrospinal fluid (CSF) is a valuable source of biomarkers for neurological diseases, but detection of low-abundance proteins is often masked by highly abundant proteins. Enrichment strategies can enhance proteomic coverage and improve biomarker discovery, yet comparative evaluations of such methods in equine CSF are limited. Objective: This study compared the ProteoMiner Small-Capacity Enrichment Kit and the PreOmics Enrich-iST Kit for their ability to deplete high-abundance proteins and enhance detection of low-abundance proteins relevant to neuropathology. Methods: Equine CSF samples were...
Effect of season of collection and storage method on equine hair cortisol concentration. Serum cortisol concentration is one of the common physiological reference standards for evaluating stress in animals. However, it only evaluates acute stress at the moment of blood collection. Hair cortisol concentrations (HCC) could provide a method for assessing cortisol concentrations over extended periods of time, giving an estimate of chronic stress levels. In this case, high HCC results could help equine managers alter management practices to improve welfare. Objective: To evaluate the effect of the month of collection and the storage method on equine HCC. Methods: Tail hair was collecte...
Comparative analysis of the transcriptome and methylome in distinct trophoblast populations at the time of embryo implantation in mares. BACKGROUND: During equine implantation, two distinct trophoblast subpopulations are identified: the invasive chorionic girdle (CG) and the non-invasive allantochorion. The invasive CG trophoblast is believed to modulate the maternal immune response and facilitates embryo attachment to the endometrium, while the allantochorion contributes to placental development without breaching the endometrium. However, the mechanisms underlying differential adhesion and immune modulation by these two lineages remain poorly understood. This study aimed to investigate the regulation of biological processes un...
Competitive enzyme-linked immunosorbent assay to quantify hepcidin in horse serum. Hepcidin (Hepc) is a peptide hormone primarily produced by hepatocytes and considered the master regulator of iron homeostasis, with Hepc controlling the release of iron within the circulation. Despite the renewed interest in iron-related research due to Hepc discovery, there is limited data on serum hepcidin in horses. The objective of the study was to develop and partially validate a competitive enzyme-linked immunosorbent assay (ELISA) to quantify serum hepcidin in horses. Precision was evaluated using horse serum samples representing low, medium and high Hepc concentrations, yielding intra...
