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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.
The effect of intravenous fluids administered prior to competition: A randomized clinical. Administration of intravenous fluids prior to competition is common at major equestrian competitions, yet few studies have evaluated the benefits of this practice. Objective: The hypothesis was that pre-ride intravenous fluid therapy would be associated with a lower heart rate and improved laboratory hydration parameters during or after the ride. Methods: 14 client owned horses entered in a 45 km ride in extreme heat and terrain were randomly assigned to receive IV fluids (IVF) or no IV fluids (NIV) the day before the ride. Blood samples and physical examination findings were collected at 6 ti...
Detection of Cyclosporine in Horse Hair After Ocular Implant Administration. This study describes the detection and temporal distribution of cyclosporine in equine hair following ocular implant administration in a Thoroughbred gelding. Mane hair samples were collected at 172- and 217-day post-implantation and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after alkaline hydrolysis and liquid-liquid extraction. The method achieved an estimated limit of detection of 0.1 pg/mg. Cyclosporine was detected in hair at both sampling time points, with segmental analysis revealing distribution patterns consistent with systemic exposure and incorporation ...
Effects of quercetin supplementation in cooling extender on sperm quality of stallions. Oxidative stress from excessive reactive oxygen species (ROS) during cooling impairs equine sperm quality. Quercetin, a flavonoid with antioxidant properties, has been proposed to reduce oxidative damage in cooled semen. Objective: This study aimed to identify a suitable quercetin concentration among those tested for cooled equine semen and evaluate its effects on sperm kinematics, membrane integrity, and oxidative stress parameters. Methods: Four experiments were conducted. Experiment 1 (n = 8 ejaculates, 4 stallions) evaluated quercetin at 0, 20, 30, or 45 µg/mL at 5°C for 24 h. Experiment...
Clinical outcomes of a novel flowable calcium silicate cement in equine endodontic treatment. Orthograde endodontic treatment of equine cheek teeth is performed infrequently with variable success rates of obturation with calcium hydroxide paste (CHP). A novel flowable calcium silicate cement (eCSC) formulated for use as an obturation material in equine teeth has demonstrated acceptable canal filling volumes and evidence of dentinal tubule penetration. However, clinical outcomes of eCSC-obturated equine teeth have not been reported. Objective: To report the clinical outcomes of orthograde endodontic treatment using eCSC. Methods: Retrospective case series. Methods: Horses that underwent...
Detection and confirmation of hypoxia-inducible factor activating agents in equine plasma and urine by liquid chromatography coupled to mass spectrometry. Hypoxia-inducible factor (HIF) activating agents can promote the transcription of the erythropoietin gene, enhancing an athlete's performance, and are therefore prohibited in sports. A liquid chromatography coupled to high-resolution mass spectrometry (LCHRMS) method was developed for the detection and confirmation of 11 HIF-activating substances in equine plasma and urine. Ammonium formate added to the mobile phase system suppressed the responses of all the analytes in either positive or negative ion mode and thus was not used. The starting organic percentage in the mobile phase gradient and ...
Isolation, Expansion and Characterization of Mesenchymal Stromal Cells from Fresh and Cryopreserved Umbilical Cord Tissues in Horse and Dog. Mesenchymal stromal cells (MSCs) derived from perinatal tissues represent a promising resource for regenerative medicine due to their proliferative, immunomodulatory, and differentiation properties. Among these tissues, the umbilical cord is considered an accessible and ethically acceptable source of MSCs. This study evaluated the feasibility of whole umbilical cord cryopreservation in canine and equine species and assessed the biological properties of MSCs isolated from cryopreserved tissues compared with fresh samples. Equine Wharton's jelly and canine umbilical cord tissues were processed e...
Intrauterine ozone therapy reduces bacterial load in mares with mixed bacterial endometritis: a longitudinal study. Mixed bacterial endometritis poses a therapeutic challenge in mares due to the coexistence of multiple pathogens with divergent antimicrobial susceptibility profiles. This study evaluated the effects of intrauterine ozone insufflation on uterine bacterial load and endometrial inflammatory response in mares with naturally acquired mixed bacterial endometritis. Fifteen mares with mixed bacterial infection were allocated to two treatment groups: a pathological control group receiving pure medical oxygen insufflation (PC; n = 6) and an ozone-treated group receiving 42 µg/mL ozone for three c...
Approaches to Thoroughbred Horse Identification in Diverse Sample Types Using Multiplexed SNP-PCR Sequencing Panels. The ability to determine the identity of equine samples would be highly advantageous in cases of ambiguous sample source, as individual-specific controls for gene editing detection, or forensic examination of suspect biological materials in potential doping cases. To this end, a SNP-PCR sequencing-based panel comprising 93 markers in one reaction was designed and investigated for use in Thoroughbred horses. Marker amplification performance and discriminating power were calculated against a population survey of 264 Thoroughbred whole blood samples from routine doping testing. The sensitivity of...
The in vitro application of high-intensity laser therapy on equine platelet-rich plasma and autologous protein solution does not alter select cytokine and growth factor concentrations. To quantify the effects of high-intensity laser therapy (HILT) on the concentration of key immunomodulatory cytokines and growth factors in equine platelet-rich plasma (PRP) and autologous protein solution (APS). We hypothesized that HILT treatment of PRP and APS in vitro would increase the concentration of cytokines and growth factors compared to untreated controls. Unassigned: Whole blood was collected from 6 adult horses housed at the University of Pennsylvania in March 2025 and processed into PRP or APS using commercially available systems (Zoetis). Platelet-rich plasma and APS samples wer...
Combined effects of pre-freeze cholesterol-loaded cyclodextrin and post-thaw pentoxifylline on the functional longevity of cryopreserved stallion sperm. Cryopreservation causes structural and metabolic damage to stallion spermatozoa, impairing post-thaw functional competence. Objective: This study evaluated whether cholesterol-loaded cyclodextrin (CLC) supplementation before freezing, combined with pentoxifylline (PTX) after thawing, improves the functional quality, oxidative status, mitochondrial activity, and preliminary fertility outcomes of cryopreserved stallion semen. Methods: Semen from twelve stallions was assigned to four CLC concentrations (0, 1, 1.5, and 2 mg), frozen, thawed, and divided into subgroups with or without PTX supplemen...
Effects of a dimethylformamide-glycerol combination on freezing behavior, post-thaw sperm quality, and sperm-oocyte interaction in Arabian stallions. Although glycerol (GLY) and dimethylformamide (DMF) are widely used cryoprotectant agents (CPAs) for freezing equine semen, their combined use remains less characterized. Objective: To evaluate the effects of DMF, GLY, and their combination (DMF+GLY) on freezing behavior, sperm kinematics, membrane integrity, and IVF-related functional endpoints in Arabian stallion sperm. Methods: Twenty-four ejaculates from four stallions (six/male) were diluted in BotuSemen-Gold® extender supplemented with 5% DMF, 5% GLY, or 3% DMF+ 3% GLY, packed into 0.5-ml straws, and frozen. Sperm-oocyte binding and pro...
SURVIVAL ON THE MARGINS: NICHE PARTITIONING AND DENSITY-DEPENDENT SPATIAL DISTRIBUTION OF EQUINE CYATHOSTOMINS. Habitat preferences of gastrointestinal nematodes are well-documented in small hosts, but equine cyathostomin diversity has largely been characterized only at farm or regional levels. While organ-level site preferences are known, within-organ spatial distributions remain unexplored. We investigated the longitudinal and radial distribution of adult cyathostomins within the large-intestinal lumen. Intact large intestines from 5 Shetland ponies were frozen post-necropsy and transected into 4-cm sections. Three micro-habitat samples (2.5 cm diameter) were collected per section. We recovered 2,444 ...
Plasma Homocysteine Concentrations in Horses with Left-Sided Valvular Heart Disease. In human medicine, homocysteine, a sulfur-containing amino acid, is used as a biomarker reflecting endothelial dysfunction, inflammation, thrombogenesis, oxidative stress and cardiovascular disease. An automated enzyme-cycling assay for homocysteine, established for humans, has been validated and reference ranges have been created for horses. In this study the homocysteine assay was further evaluated by assessing short-term stability (whole blood and plasma samples stored at 4 °C and room temperature for 24 h), long-term stability (two years), the effect of different anticoagulants (lithium h...
Effects of equine sperm incubation under capacitation-supporting conditions on homologous and heterologous ICSI outcomes. Intracytoplasmic sperm injection (ICSI) in horses has become sufficiently efficient to be considered for clinical and commercial application; however, several factors influencing its efficiency remain poorly understood. This study evaluated whether incubating equine sperm under capacitating conditions induces capacitation-associated changes and affects embryo development following homologous equine ICSI, as well as oocyte activation, early cleavage, and 5-methylcytosine (5 mC) and 5-hydroxymethylcytosine (5hmC) levels following heterologous ICSI using bovine oocytes. Equine spermatozoa were ...
Molecular detection of African horse sickness virus from selected areas in Ethiopia. Equines are vital to global economies and support millions of livelihoods. However, their productivity and welfare are severely hindered by infectious diseases such as African Horse Sickness, which causes major socio-economic losses in Ethiopia. A cross-sectional study was conducted from November 2022 to May 2023 to isolate and detect African horse sickness virus in equines across epidemic areas of Ethiopia. In total, 30 blood samples and 2 tissue representative specimens were aseptically collected from clinically sick and recently dead animals and transported under cold chain to the National ...
The effect of fenugreek (Trigonella foenum-graecum) on stallion spermatozoa motility and vitality in vitro. This study evaluated the effects of Trigonella foenum-graecum L. (fenugreek) seed extract on stallion spermatozoa quality in vitro, focusing on motility, vitality, and functional competence. In vitro spermatozoa analyses showed that fenugreek seed extract did not demonstrate overt cytotoxicity under the tested conditions: S1 = 1562 µg/mL; S2 = 781 µg/mL; S3 = 390.5 µg/mL; S4 = 195.25 µg/mL; S5 = 97.62 µg/mL; S6 = 48.81 µg/mL; S7 = 24.40 µg/mL. Computer-assisted semen analysis (CASA) revealed a concentration- and time-dependent Treatment × Time interac...
Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantification of twenty-five steroids in equine serum. Steroids are potential biomarkers for monitoring equine pregnancy. However, immunoassays currently used for their quantification suffer from cross-reactivity and limited specificity, thus requiring more accurate methods. This study reports the development and validation of a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of 25 steroids covering the main biosynthetic pathways of progestogens, corticosteroids, androgens, and estrogens. Steroids were extracted by protein precipitation followed by evaporation, derivatization, and reconstitut...
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...
Density gradient centrifugation preserves sperm kinetics and morphology in stallion ejaculates with severe urospermia. Urospermia is a frequent cause of subfertility in stallions due to the rapid deterioration of sperm quality. Objective: This study aimed to evaluate the effectiveness of different processing methods in minimizing the deleterious effects of urine on cooled equine semen. Methods: Ejaculates from 12 stallions were subjected to simple dilution, conventional centrifugation, and colloidal density gradient centrifugation, under challenges of 0%, 20%, and 50% urine, and stored at 5 °C for 24 h. Results: A severe dose-dependent effect was observed: increasing urinary concentration progressively reduce...
Clinical and microbiological evaluation of a phytotherapeutic intrauterine therapy in mares with bacterial endometritis: a preliminary study. Endometritis is a major cause of subfertility in mares and an important source of economic loss in the equine industry. Growing concerns regarding antimicrobial resistance have stimulated interest in alternative therapies. Objective: To evaluate the clinical efficacy of a phytotherapeutic intrauterine treatment in mares with endometritis and its in vitro antimicrobial activity against uterine isolates. Methods: Twenty mares with bacterial endometritis based on uterine culture, cytology, and ultrasonography were randomly assigned to two groups (n = 10). Control mares received uterine lavage fol...
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...
