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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.
Evaluation of a new immunoassay for measuring equine serum insulin and plasma adrenocorticotrophic hormone: A method comparison study. Insulin dysregulation (ID) and pituitary pars intermedia dysfunction (PPID) are common equine endocrinopathies detected by measurement of circulating insulin and adrenocorticotrophic hormone (ACTH) concentrations. Different assays may yield variable results, limiting comparability. Objective: To compare immunoassays developed specifically to measure equine insulin (VIDAS® Equine Insulin; enzyme-linked fluorescent assay [ELFA]) and equine ACTH (VIDAS® Equine ACTH; ELFA) with results obtained from a chemiluminescence immunoassay (CLIA) measured using IMMULITE 2000XPi, a chemiluminescence enzym...
Evaluation of mosquito transmission of equine hepacivirus in laboratory and field studies. Equine hepacivirus (EqHV) is a hepatotropic virus, closely related to human hepatitis C virus (HCV), that can cause acute or chronic infection in horses. As with HCV, transmission is thought to occur primarily through parenteral, particularly blood-borne, routes. Vertical transmission from mare to foal has been reported but appears to be uncommon. The high seroprevalence in horse populations and the occurrence of infection in isolated horses indicate efficient horizontal transmission routes exist. One such possibility is biological or mechanical transmission by arthropods, such as mosquitoes, ...
Blood pressure, electrolyte and fluid balance in horses with insulin dysregulation after short-term treatment with canagliflozin. Short-term treatment with SGLT-2 inhibitors decreases blood pressure in people with type 2 diabetes. The aim was to investigate the impact of a 3-week treatment with canagliflozin on blood pressure in insulin dysregulated (ID) horses and to study effects on fluid balance. This was a single-centre, randomised, double-blind, placebo-controlled, parallel design study. The primary study evaluated glucose and insulin responses during an oral sugar test, and these results have been published previously. Here we report the effect of canagliflozin treatment on blood pressure, PCV, serum proteins and e...
Synthesis and Characterization of 2-(1-Hydroxyethyl)Promazine Sulfoxide-d4 HCl as an Internal Standard for the Accurate Quantitation of 2-(1-Hydroxyethyl)Promazine Sulfoxide in Equine Urine. Acetylpromazine, 1-{10-[3-(dimethylamino)propyl]-10H-phenothiazin-2-yl}ethenone, CHNOS, 326.46 g·mol is a phenothiazine derivative at one time used in human medicine as an antipsychotic medication but now predominantly used in veterinary medicine as a sedative/tranquilizer and referred to as acepromazine. In performance horses its use is regulated by using a 10 ng/mL threshold for the major urinary metabolite 2-(1-hydroxyethyl) promazine-sulfoxide (HEPS) in equine urine. To enable accurate quantitation of HEPS in equine urine we have synthesized and purified hydroxyethylpromazine sulfoxid...
Centrifugation of peritoneal fluid improves visual assessment of strangulating lesions in equine colic. Early and accurate recognition of a strangulating lesion is essential to expedite surgical intervention in horses presenting with colic. Objective: To evaluate the use of a colour grading scale in the visual assessment of peritoneal fluid, before and after centrifugation, and assess its ability to predict the presence of a strangulating lesion. Methods: Prospective clinical study. Methods: Peritoneal fluid samples from 231 equids presenting with colic were visually analysed immediately after collection and assigned a numerical value based on a developed colour scale. Peritoneal fluid was centr...
Evaluating ex vivo toll-like receptor agonist response as a potential biomarker in equine gastric ulcer syndrome. Equine gastric ulcer syndrome (EGUS) is highly prevalent in horses. Despite its presumed painful nature, evaluation of objective biomarkers associated with EGUS presence and severity remains limited. The objective of this study was to investigate the potential of interleukin-1 beta (IL-1β) release by peripheral blood mononuclear cells (PBMCs) following ex vivo stimulation of toll-like receptors 2 and 4 (TLR2 and TLR4) as a potential biomarker for EGUS status, with gastric lesions used as a proxy for presumed EGUS-associated pain. Venous blood was collected from 77 horses, PBMCs were isolated ...
Effect of sample storage on pH-value in equine faeces. Measurements of pH-value in equine faeces are often performed in studies on nutrition and health in horses, but a standard protocol for it is lacking despite the knowledge that different sample handling may affect the pH-value. Sample handling prior to pH-measurement is often determined by practical settings and has been reported to vary in published studies within the area. The purpose of the current study was therefore to investigate the influence of different combinations of storage time and temperature on the pH-value in equine faecal samples in comparison to immediate pH-measurement at de...
Preliminary evaluation of beta-endorphin alone and in combination with adrenocorticotropic hormone for the diagnosis of pituitary pars intermedia dysfunction in equids. Limited published data exist regarding the diagnostic utility of beta-endorphin (β-END) for pituitary pars intermedia dysfunction (PPID), either compared with or in combination with adrenocorticotropic hormone (ACTH). Objective: To evaluate β-END as a potential diagnostic test for PPID in comparison with ACTH and to explore the diagnostic performance of combined tests. Methods: Ninety-eight equids aged 19-32 years, owned by the foundation "Stiftung für das Pferd," were included. Methods: A cross-sectional study was conducted in January 2025. Haircoat abnormalities were scored as absent, mi...
Microbiological, Molecular Diagnosis and Antimicrobial Resistance of Bordetella bronchiseptica in the Equine Reproductive Tract in Brazil. The genus Bordetella comprises 16 species of aerobic Gram-negative bacteria, with the most notable being B. pertussis, B. parapertussis and B. bronchiseptica. The latter stands out for its ability to infect various hosts, causing both respiratory and reproductive issues, such as endometritis and infertility in mares. These infections impair reproductive efficiency and facilitate the spread of the pathogen among susceptible animals. Objective: The present study aimed to report the isolation and molecular diagnosis of B. bronchiseptica associated with endometritis and infertility in mares in Bra...
Comparative analysis of the karyotypes of Equus caballus and Equus asinus in the context of contemporary genomic data. Karyotypes of species within the genus Equus display considerable variation in chromosome number and are characterised by numerous structural rearrangements that have played a pivotal role in their evolutionary divergence and speciation. Equus caballus (horse), 2n = 64 and Equus asinus (donkey), 2n = 62, constitute particularly interesting models for comparative studies due to the diversified morphology of their chromosomes, including acrocentric and submetacentric pairs, documented Robertsonian translocations, and centric fusions. In recent decades, widely applied molecular cytogenetic techni...
Effects of pulsed electromagnetic therapy on the major hematological and biochemical parameters in horses. Pulsed electromagnetic field (PEMF) therapy, a commonly used supplementary treatment in the horse industry, shows promise in enhancing circulation, reducing inflammation, and supporting cellular repair; however, its effects on equine physiology remain unclear. Limited research and inconsistent protocols create uncertainty about its efficacy and safety in horses. Our study evaluates the short-term effects of PEMF on hematological and biochemical parameters in horses. Five active sport horses underwent PEMF therapy for five consecutive days using a full-body blanket at specific frequencies. Bloo...
Correlation between microbial communities, metabolites, and bioactivities in fermented mare’s milk revealed by metagenomics and metabolomics. Koumiss exhibits various functional properties, including antioxidant, hypoglycemic, and antihypertensive effects; however, natural fermentation is characterized by an uncontrollable microbial community structure, significant fluctuations in product quality, and a lack of specialized fermentation agents. The mechanisms underlying the links between microbial communities, functional metabolites, and bioactivity in natural and inoculated fermentation systems remain unclear. Therefore, this study employed bioactivity assays, metagenomics, and non-targeted metabolomics to systematically analyze dif...
Evaluation of a commercial multiplex immunoassay for assessing uterine inflammation in mares susceptible to persistent breeding-induced endometritis. Persistent breeding-induced endometritis (PBIE) is characterized by a prolonged and dysregulated uterine inflammatory response following breeding. Although inflammatory cytokines are central to the post-breeding immune response, their assessment in the equine uterus has historically relied largely on gene expression analyses. Multiplex immunoassays provide an opportunity to simultaneously quantify multiple cytokine proteins within uterine secretions; however, the application of equine-specific multiplex immunoassays utilizing the Luminex platform to low-volume uterine lavage (LVL) fluid remain...
Effects of the Freezing Diluent Tonicity and Prefreezing Cooling Rates on Post-Thaw Sperm Quality Markers in the Pure-Bred Arabian Stallion. Stallion semen cryopreservation remains limited by inconsistent post-thaw sperm quality and fertility. This study evaluated the effects of freezing extender effective tonicity and prefreezing cooling rate on post-thaw sperm characteristics in pure-bred Arabian stallions. In a two-phase design, ejaculates ( = 40) from 10 stallions were processed using extenders with different osmotic conditions (330, 390, and 450 mOsm/kg) and subjected to cooling durations of 45 min, 1.5 h, or 3 h. Sperm quality was assessed using CASA, functional assays, flow cytometry, transcriptomic analysis, and...
Energy Metabolism Dysregulation in Horses and Foals. Dysregulation of energy metabolism occurs in horses and foals with transient systemic illness, malnutrition, or chronic metabolic diseases. The pathophysiologic processes resulting in energy dysregulation are important to interpret, understand, and appropriately monitor laboratory abnormalities associated with various disease states (hypoglycemia and hyperglycemia), as well as to aid in the management of energy dysregulation through nutritional and medical management.
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 ...
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...
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...
