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Topic:Laboratory Methods
Multiparametric Flow Cytometry for Determination of Viability, Caspase 3 and 7 Activity, and Lipid Peroxidation Adduct (4-Hydroxynonenal) in Equine Spermatozoa. Flow cytometry is a powerful tool for the analysis of cell samples formed of multipopulations, such as spermatozoa. In recent years, multiparametric cytometers have evolved, allowing the study of different cellular characteristics, such as protein expression, DNA analysis, or mitochondrial activity. Whether using traditional fluorescent dyes or fluorophore-conjugated antibodies, each cell or cellular component is individually stained, the sample is analyzed at high velocities, and then is displayed and interpreted in a dot-plot. We hereby describe the procedure to perform a multiparametric flo...
Visualizing neutrophil extracellular traps in septic equine synovial and peritoneal fluid samples using immunofluorescence microscopy. Septic synovitis and peritonitis are routinely diagnosed in horses based on clinical examination findings and laboratory assessment of synoviocentesis and abdominocentesis samples, respectively. Diagnosis is difficult in some cases because of an overlap in laboratory results for septic and non-septic inflammation. Neutrophil extracellular trap (NET) formation is part of the innate immune response against pathogens. Identifying and quantifying NETs, which have not been explored in clinical samples from horses with septic synovitis and peritonitis, to our knowledge, may be helpful in detecting i...
Comparison of incisional bursting strength of a bidirectional absorbable knotless suture material versus a standard continuous absorbable suture material for closure of the equine linea alba. To evaluate the use of bidirectional knotless barbed suture material for closure of the equine linea alba. Methods: Laboratory study. Methods: Adult light horse cadavers (n = 24). Methods: A 25 cm incision was made through the linea alba, and a 200 L polyurethane bladder was positioned within the abdomen. The linea alba was closed either using USP2/EP5 bidirectional polydioxanone barbed suture or USP2/EP5 standard polydioxanone suture in a simple continuous pattern. Closure time was recorded for each suture type. The bladder was air-insufflated at 40 L/min, and the pressure at body w...
Rotation of liquid-preserved artificial insemination doses on roller benches affects sperm quality during storage in stallions. Appropriate stallion semen handling is of great importance in equine artificial insemination (AI) industry. Optimal treatment of AI doses is aiming for best sperm preservation by excluding strong environmental influences and adding media that favour sperm survival. One method widely used in stallion sperm handling is the rotation of liquid-preserved semen samples on roller benches during storage. As previous studies in boars give rise to the fact that rotation should not be considered beneficial for spermatozoa anymore, the present study investigated the influence of continuous rotation of dil...
Flow cytometric detection and identification of different leukocyte subpopulations in stallion semen. To improve accuracy in evaluating stallion ejaculates, an antibody-based, flow cytometric assay for the detection and identification of leukocyte subpopulations (CD4-, CD8-, CD21-, CD172a-positive cells) in stallion semen (n = 12) was established. For establishment of the assay, native semen was supplemented with blood leukocytes (control: 20% leukocytes, 80% sperm cells) and analysed by flow cytometry. Adding antioxidants (ascorbic acid and butylated hydroxytoluol) to semen immediately after collection inhibited rapid death of lymphoid cells in sperm leukocyte mixtures. In control set-ups...
An extensive screening method for the identification and quantitation of ecdysteroids in equine urine and plasma using liquid chromatography coupled with mass spectrometry. Recently, there has been a report suggesting that ecdysteroids can enhance sports performance, making them relevant substances in doping control. Hence, it is imperative to examine the analytical characteristics of ecdysteroids in biological samples to identify their misuse in competitive sports. Methods: To assess the doping of ecdysteroids such as ecdysone, ecdysterone, ponasterone A, turkesterone, and ajugasterone C, a fast and sensitive extraction and detection method was developed, optimized, and validated using equine urine and plasma. Different extraction techniques, namely, solid-phase...
Effects of insulin-like growth factor-1 on the proliferation and apoptosis of stallion testicular cells under normal and heat stress culture conditions. This study investigated the effect of heat stress on stallion testicular cells (TCs) and the effect of insulin-like growth factor (IGF)-1 on TC viability, proliferation, and apoptosis, including different stages of germ cells. TCs were divided into control or treatment groups with 0.01, 0.1, 1, 10, and 100 ng/mL of recombinant human IGF-1 (rhIGF-1) for 24 h at 34 °C and 37 °C. The population and viability were measured before and after treatment. The effects of rhIGF-1 on TC viability, proliferation, and apoptosis were determined using RT-qPCR. Proliferating cell nuclear antigen (PCNA) a...
Determination of optimal storage time and temperature for the detection of red blood cell and platelet surface-associated immunoglobulin by flow cytometry in healthy horses. Differentiating immune-mediated causes from other causes of anemia and thrombocytopenia can be challenging. Flow cytometry can detect surface-associated immunoglobulin (sIg) on red blood cells (RBC) and platelets (PLT) in dogs and horses. Sample storage parameters for ideal assay performance has not been evaluated in horses. The study objective is to identify optimal storage time and temperature of equine whole blood for the detection of RBC-sIg and PLT-sIg via flow cytometry. Both assays were performed on samples at time 0, 4, 24, 48, and 72 h post collection. RBC-sIg samples were stored at ...
Identification of allergens in Artocarpus heterophyllus, Moringa oleifera, Trianthema portulacastrum and Syzygium samarangense. It is clinically important to identify allergens in Artocarpus heterophyllus (jackfruit), Moringa oleifera (moringa), Trianthema portulacastrum (horse purslane) and Syzygium samarangense (rose apple). This study included 7 patients who developed anaphylaxis to jackfruit (1), moringa (2), horse purslane (3) and rose apple (1). We sought to determine allergens in the edible ripening stages of jackfruit (tender, mature, and ripened jackfruit) and seeds, edible parts of moringa (seeds, seedpod, flesh inside seedpod, and leaves), horse purslane leaves and ripened rose apple fruit. The persistence o...
Preliminary evaluation of reference intervals for a point-of-care viscoelastic coagulation monitor (VCM Vet) in healthy adult horses. To evaluate a point-of-care viscoelastic coagulation monitor (VCM Vet) for use in horses by assessing variability between devices and establish reference intervals (RIs) for healthy adult horses. Methods: Prospective observational study. Methods: Two university teaching hospitals. Methods: Healthy adult horses (n = 68). Methods: None. Results: Blood collected by direct jugular venipuncture was applied directly from the syringe into 2 VCM Vet cassettes to establish coefficients of variation (CVs) and RIs for reported parameters of clotting time (CT), clot formation time (CFT), alpha angle, am...
Validation of a serum ELISA test for cyathostomin infection in equines. Cyathostomins are ubiquitous equine nematodes. Infection can result in larval cyathostominosis due to mass larval emergence. Although faecal egg count (FEC) tests provide estimates of egg shedding, these correlate poorly with burden and provide no information on mucosal/luminal larvae. Previous studies describe a serum IgG(T)-based ELISA (CT3) that exhibits utility for detection of mucosal/luminal cyathostomins. Here, this ELISA is optimised/validated for commercial application using sera from horses for which burden data were available. Optimisation included addition of total IgG-based calibr...
Effect of dipotassium-ethylenediaminetetraacetic acid or lithium-heparin treatments and storage times on selected clinicopathologic analytes in equine synovial fluid. Sample processing methods and storage time affect the outcome of biochemical analysis. Objectives: We aimed to assess the effects of dipotassium-ethylenediaminetetraacetic acid (K2-EDTA) and lithium-heparin treatments and storage times on selected analytes in equine synovial fluid (SF). Conclusions: The analytes-except for TP-became unstable within a few hours postcollection. Lithium-heparin and K2-EDTA treatments significantly altered ALP, LDH, TNCCs, and pH but not the TP concentrations of equine SF. Studies establishing reference intervals for these analytes based on the anticoagulant used ...
Performance of a manually operated salad spinner centrifuge for serum separation in the healthy domestic horse (Equus caballus) and southern white rhinoceros (Ceratotherium simum). Field veterinarians and researchers studying wild species, such as the southern white rhinoceros, often work in remote areas with limited access to standard laboratory equipment, hindering the ability to measure serum analytes.
Objectives: The first objective was to produce an inexpensive, manually operated centrifuge that could accept standard laboratory tubes by modifying a consumer-grade salad spinner with low-cost materials. The second objective was to compare biochemistry analysis results obtained from equine and southern white rhinoceros serum separated by traditional laboratory and m...
Standardization of a Sex-Sorting Protocol for Stallion Spermatozoa by Means of Absolute RT-qPCR. Sperm sexing is a technology that can generate great economic benefits in the animal production sector. Techniques such as sex-sorting promise over 90% accuracy in sperm sexing. However, for the correct standardization of the technique, some laboratory methodologies are required. The present manuscript describes in detail a standardized equine sperm sex-sorting protocol using an absolute qPCR-based methodology. Furthermore, the results of absolute qPCR were implemented and validated by generating equine/bovine heterologous embryos by intracytoplasmic sperm injection (ICSI) of presumably sexed ...
Quadruplex Real-Time TaqMan® RT-qPCR Assay for Differentiation of Equine Group A and B Rotaviruses and Identification of Group A G3 and G14 Genotypes. Equine rotavirus A (ERVA) is the leading cause of diarrhea in foals, with G3P[12] and G14P[12] genotypes being the most prevalent. Recently, equine G3-like RVA was recognized as an emerging infection in children, and a group B equine rotavirus (ERVB) was identified as an emergent cause of foal diarrhea in the US. Thus, there is a need to adapt molecular diagnostic tools for improved detection and surveillance to identify emerging strains, understand their molecular epidemiology, and inform future vaccine development. We developed a quadruplex TaqMan RT-qPCR assay for differentiation of ERVA an...
Detection of Viable Streptococcus equi equi Using Propidium Monoazide Polymerase Chain Reaction. There is debate around the clinical significance of Streptococcus equi subsp. equi detection in low numbers using quantitative real-time PCR (qPCR). Propidium monoazide (PMA) qPCR has been used to differentiate DNA from viable and nonviable bacterial cells. The aim of this study was to evaluate the ability of PMA eqbE SEQ2190 triplex qPCR to differentiate DNA from viable and nonviable S. equi in positive and suspect positive clinical specimens. Fifty-seven stored (frozen and refrigerated) positive (36) or suspect positive (21) clinical specimens (determined via SeeI qPCR as the gold standard) ...
Equine synovial sepsis laboratory submissions yield a low rate of positive bacterial culture and a high prevalence of antimicrobial resistance. To investigate (1) variables associated with the likelihood of obtaining a positive culture, (2) commonly isolated microorganisms, and (3) antimicrobial resistance patterns of isolates from horses with presumptive synovial sepsis. Methods: Synovial fluid, synovium, and bone samples from equine cases with presumptive synovial sepsis submitted to the Cornell University Animal Health Diagnostic Center from 2000 to 2020 for microbial culture and antimicrobial sensitivity testing. Methods: Univariable and multivariable analyses were performed to determine the effect of variables on the likelihood o...
A hydrophobic deep eutectic solvent-based vortex-assisted liquid-liquid microextraction applied for doping control of aromatase inhibitors from equine urine. Aromatase inhibitors (AIs) can indirectly cause increased testosterone in animals, which leads to the improvement of the athletic ability of horses. For the protection of horses and the consideration of fair competition, AIs were listed as prohibited drugs by the Federation Equestre Internationale (FEI). There were several disadvantages using traditional pretreatment methods before analyzing these drugs from biological samples. A rapid and green pretreatment method has been developed by utilizing the hydrophobic deep eutectic solvent (DES)-based vortex-assisted liquid-liquid microextraction (D...
A distinct variant of the SzM protein of Streptococcus equi subsp. zooepidemicus recruits C1q independent of IgG binding and inhibits activation of the classical complement pathway. Streptococcus equi subsp. zooepidemicus (SEZ) is a major equine pathogen that causes pneumonia, abortion, and polyarthritis. It can also cause invasive infections in humans. SEZ expresses the M-like protein SzM, which recruits host proteins such as fibrinogen to the bacterial surface. Equine SEZ strain C2, which binds only comparably low amounts of human fibrinogen in comparison to human SEZ strain C33, was previously shown to proliferate in equine and human blood. As the expression of SzM_C2 was necessary for survival in blood, this study investigated the working hypothesis that SzM_C2 inhibi...
Exposure levels of animal allergens, endotoxin, and β-(1,3)-glucan on a university campus of veterinary medicine. The study aimed to determine the allergen, endotoxin and β-(1,3)-glucan concentrations at various areas on a university campus of veterinary medicine. Methods: Dust samples were collected four times a year for three years using electrostatic dust collectors (EDC) at 25 different locations on a campus of veterinary medicine and in laboratories of inorganic chemistry as a control area representing animal-free environment. Major animal allergens from dog, cat, horse, cattle and mouse, domestic mite (DM) allergens, and β-(1,3)-glucan were measured using enzyme immunoassays and endotoxin using th...
Agreement between hematocrit values determined by the Cobas b121 blood gas analyzer and the microhematocrit method in dogs, cats, and horses. Packed cell volume (PCV) is important for assessing a patient's health status. Some blood gas analyzers measure hematocrit, and the agreement with PCV varies among different analyzers. Objective: We aimed to determine the agreement between PCV measured by microcentrifugation and hematocrit measured by the Cobas b121 blood gas analyzer in dogs, cats, and horses. Methods: Whole blood samples for PCV and blood gas analysis were collected in lithium-heparin syringes and analyzed within 10 min of collection. Agreement and association between the PCV and Cobas b121 generated hematocrit were assess...
Technical note: Cartilage imaging with sub-cellular resolution using a laboratory-based phase-contrast x-ray microscope. Microscopic imaging of cartilage is a key tool for the study and development of treatments for osteoarthritis. When cellular and sub-cellular resolution is required, histology remains the gold standard approach, albeit limited by the lack of volumetric information as well as by processing artifacts. Cartilage imaging with the sub-cellular resolution has only been demonstrated in the synchrotron environment. Objective: To provide a proof-of-concept demonstration of the capability of a laboratory-based x-ray phase-contrast microscope to resolve sub-cellular features in a cartilage sample. Meth...
Application of Nanopore Sequencing for High Throughput Genotyping in Horses. Nanopore sequencing is a third-generation biopolymer sequencing technique that relies on monitoring the changes in an electrical current that occur as nucleic acids are passed through a protein nanopore. Increasing quality of reads generated by nanopore sequencing systems encourages their application in genome-wide polymorphism detection and genotyping. In this study, we employed nanopore sequencing to identify genome-wide polymorphisms in the horse genome. To reduce the size and complexity of genome fragments for sequencing in a simple and cost-efficient manner, we amplified random DNA fragme...
Screening and confirmation of recombinant human follistatin in equine plasma for doping control purposes. Recombinant human follistatin (rhFST) is a potential performance-enhancing agent owing to its stimulating effect on muscle growth. Administration of rhFST to athletes is prohibited in human sports by the World Anti-Doping Agency (WADA) and in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering published by the International Federation of Horseracing Authorities (IFHA). For effective control of the potential misuse of rhFST in flat racing, methods for screening and confirmatory analysis are required. This paper describes the development and validat...
Evaluation of the effects of medium-term (57-day) omeprazole administration and of omeprazole discontinuation on serum gastrin and serum chromogranin A concentrations in the horse. Rebound gastric hyperacidity (RGH) secondary to hypergastrinemia has been suggested to contribute to the rapid recurrence of equine squamous gastric disease (ESGD) in horses after discontinuation of omeprazole. Objective: To evaluate changes in serum gastrin and chromogranin A (CgA) concentrations in response to medium-term (57-day) omeprazole treatment and after omeprazole discontinuation. Methods: Fourteen mature Thoroughbred racehorses in simulated race training. Methods: Horses received 2.28 g of oral omeprazole PO q24h for 57 days within a 61-day period, excluding a withholding period...
Applicability of 2,4-dinitrophenylhydrazine (DNPH) method of protein oxidative damage measurement in the seminal plasma of canine ( Canis lupus familiaris) and stallion ( Equus caballus). Seminal plasma (SP) proteins are responsible for sperm functional quality. Developing a reliable method to determine the degree of oxidative damage of these proteins is important for establishing semen fertilizing ability. The main aim of the study was to verify the applicability of protein carbonyl derivatives measurement in the SP of canine and stallion, using a method with 2,4-dinitrophenylhydrazine (DNPH). The research material consisted of ejaculates obtained from eight English Springer Spaniels, and from seven half-blood stallions during the breeding and non-breeding season. The content ...
Simultaneous Determination of Pyrethrins, Pyrethroids, and Piperonyl Butoxide in Animal Feeds by Liquid Chromatography-Tandem Mass Spectrometry. The presence of insecticides like pyrethrins and synthetic pyrethroids, combined with the synergist piperonyl butoxide, in animal feeds can pose a risk to both animal and human health by contaminating the food chain. In this study, a simple and fast method was developed for the simultaneous determination of these compounds in contaminated animal feeds using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample preparation was carried out using a QuEChERS-based approach, and the method was validated with acceptable accuracy ranging from 84 to 115% and precision below 10%. The limit ...
Genomic detection of Coxiellaburnetii based on plasmid genes in horses. Q fever is a worldwide zoonosis caused by an obligate intra-cellular pathogen called affecting a broad range of animal hosts including horses. Most of the isolates found carry plasmids which genetic studies of strains suggest a critical role in survival. The correlation between an isolated plasmid type and the chronic or acute nature of the disease has always been controversial. This study was conducted to investigate the prevalence of and plasmids in horses and assess the potential role of these species as reservoirs of infection and transmission. Nested-polymerase chain reaction (PCR) a...
Culture Media Supplemented With 10% Equine Serum Provided Chondroprotection in an In Vitro Co-Culture of Cartilage and Synovial Membrane. No studies have evaluated the effect of culture in serum-free media (SF) vs. media supplemented with equine serum (ES) on co-culture of synovial membrane and cartilage tissue explants. The study objective was to evaluate the effects of equine serum supplementation on induced production of inflammatory and catabolic mediators from articular cartilage and synovial explants while in co-culture. Articular cartilage and synovial membrane explants were harvested from femoropatellar joints of five adult horses. Cartilage and synovial explants were harvested from the stifle of five horses, placed in c...
In vitro antibacterial efficacy of autologous conditioned plasma and amniotic membrane eye drops. To determine the in vitro antibacterial efficacy of equine and canine autologous conditioned plasma (ACP) and amniotic membrane extract eye drops (AMEED) against aerobic bacteria common to the corneal surface. Methods: Canine (n = 4) and equine (n = 4) anticoagulated whole blood samples were sterilely collected, pooled for each species, and processed using the Arthrex ACP® Double-Syringe System. Platelet counts were performed on ACP and pooled blood. AMEED were obtained from a commercial source. An electronic medical records search (2013-2022) identified aerobic bacteria cultured from...
