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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.
Challenges in using serological methods to explore historical transmission risk of Chlamydia psittaci in a workforce with high exposure to equine chlamydiosis. This report describes the challenges encountered in using serological methods to study the historical transmission risk of from horses to humans. In 2017, serology and risk factor questionnaire data from a group of individuals, whose occupations involved close contact with horses, were collected to assess the seroprevalence of antibodies to and identify risk factors associated with previous exposure. 147 participants were enrolled in the study, provided blood samples, and completed a questionnaire. On ELISA testing, antibodies to the Chlamydia genus were detected in samples from 17 participa...
Expression Profile of the Chromosome 14 MicroRNA Cluster (C14MC) Ortholog in Equine Maternal Circulation throughout Pregnancy and Its Potential Implications. Equine chromosome 24 microRNA cluster (C24MC), the ortholog of human C14MC, is a pregnancy-related miRNA cluster. This cluster is believed to be implicated in embryonic, fetal, and placental development. The current study aimed to characterize the expression profile of this cluster in maternal circulation throughout equine gestation. The expression profile of miRNAs belonging to this cluster was analyzed in the serum of non-pregnant (diestrus), pregnant (25 d, 45 d, 4 mo, 6 mo, 10 mo), and postpartum mares. Among the miRNAs examined, 11 miRNAs were differentially expressed across the analyzed ...
Secretion of equine chorionic gonadotropin and its association with supplementary corpus luteum formation and progesterone concentration in Hokkaido native pony recipient mares. The objectives of this study were to determine the plasma profile of equine chorionic gonadotropin (eCG) and its association with the formation of supplementary corpus luteum (CL) and plasma progesterone concentrations in embryo transfer Hokkaido native pony recipient mares. Blood samples and transrectal ultrasound examination of the reproductive tract were carried out weekly from the day of ovulation until week 32 of gestation (n = 4). Plasma concentrations of eCG and progesterone were measured by enzyme immunoassays. The eCG concentration was first detectable at week 5 for 2 mares and at wee...
Clostridioides (Clostridium) Difficile in Food-Producing Animals, Horses and Household Pets: A Comprehensive Review. is ubiquitous in the environment and is also considered as a bacterium of great importance in diarrhea-associated disease for humans and different animal species. Food animals and household pets are frequently found positive for toxigenic without exposing clinical signs of infection. Humans and animals share common ribotypes (RTs) suggesting potential zoonotic transmission. However, the role of animals for the development of human infection due to remains unclear. One major public health issue is the existence of asymptomatic animals that carry and shed the bacterium to the environment, an...
Salivary Scavenger and Agglutinin (SALSA) Is Expressed in Mucosal Epithelial Cells and Decreased in Bronchial Epithelium of Asthmatic Horses. The Salivary Scavenger and Agglutinin (SALSA) protein is an innate immune protein with various alleged functions, including the regulation of inflammation and tissue remodeling. Transcriptomic studies of severe equine asthma (SEA) showed downregulation of the gene encoding SALSA in bronchial epithelium of asthmatic compared to non-asthmatic horses. This study aimed to characterize expression of SALSA in equine tissues by immunohistochemistry (IHC), corroborate potential differences in epithelial gene expression between asthmatic and non-asthmatic horses, and assess the structure of equine SALS...
In vitro production of horse embryos predisposes to micronucleus formation, whereas time to blastocyst formation affects likelihood of pregnancy. Invitro embryo production is an increasingly popular means of breeding horses. However, success is limited by a high incidence of early embryo loss. Although there are various possible causes of pregnancy failure, chromosomal abnormalities, including aneuploidy, are important potential contributors. This study evaluated the frequency of micronucleus formation as a proxy for aneuploidy in invitro-produced (IVP) and invivo-derived horse blastocysts. Associations between IVP embryo morphology, frequency of nuclear abnormalities and the likelihood of pregnancy were investigated. IVP blastocysts ex...
Evaluation of multiple allergen simultaneous (sIgE) testing compared to intradermal testing in the etiological diagnosis of atopic dermatitis in horses. Although intradermal testing (IDT) is commonly used in the etiological diagnosis of allergies, in vitro testing for specific IgE (sIgE) is an attractive alternative. Currently, new laboratory techniques in veterinary allergological practice, including multiple allergen simultaneous tests (MASTs), gradually supersede in vivo tests. Both, serological (sIgE) and IDTs in fourteen atopic Malopolski horses were performed. Correlation and agreement between test results were evaluated. Receiver operating characteristic analysis showed that sIgE to had the best diagnostic performance (Area under the R...
vB_PaeM_MIJ3, a Novel Jumbo Phage Infecting Pseudomonas aeruginosa, Possesses Unusual Genomic Features. Phages are the most abundant biological entity on Earth. There are many variants in phage virion sizes, morphology, and genome sizes. Large virion sized phages, with genome sizes greater than 200 kbp have been identified and termed as Jumbo phages. These phages exhibit certain characteristics that have not been reported in phages with smaller genomes. In this work, a jumbo phage named MIJ3 (vB_PaeM_MIJ3) that infects PAO1 was isolated from an equine livery yard in Leicestershire, United Kingdom. The genome and biological characteristics of this phage have been investigated. MIJ3 is a Myovirus...
Plasma steroid profiles before and after ACTH stimulation test in healthy horses. This study describes steroid profiles in equine plasma before and after ACTH stimulation. In human medicine, other steroids have been shown to have a more pronounced reaction to an ACTH stimulation test than cortisol. This study aimed to determine if the same was true for the horse. A total of 11 clinically healthy horses were selected for this study. Ethylenediaminetetraacetic acid plasma samples were taken before and 60 min after stimulation with 1 μg/kg BW of synthetic ACTH administered intravenously. The samples were analyzed for cortisol, 11-deoxycortisol, 21-deoxycortisol, cortisone, ...
Isolation and identification of an isoflavone reducing bacterium from feces from a pregnant horse. The aim of this research was to isolate bacteria capable of biotransforming daidzein from fresh feces from pregnant horses. A Hungate anaerobic roller tube was used for anaerobic culture. Single colonies were picked at random and incubated with daidzein. High performance liquid chromatography was used to detect whether the isolated bacteria were able to biotransform the substrate. A strain capable of reducing daidzein was selected and characterized using sequence analysis of 16S rDNA, and a phylogenetic tree was constructed. The morphological physiological and biochemical characteristics of th...
The effect of counting duration on quantitative fecal egg count test performance. Fecal egg counts are the primary diagnostic tools of equine parasitology and use of the McMaster test and its variants in clinical practice is widely recommended. Manual counting is, however, prone to various sources of human error. For example, in real-world situations analysts can be under significant pressure to process high numbers of samples in a limited time. This practice could affect test result quality, but yet no studies have determined whether this is the case. This study's purpose was to assess the effect of shortened counting duration (from either restricting counting time or coun...
Establishment and characterization of proliferating primary cultures of equine epidermal keratinocytes. Skin-derived tissue cultures are a useful model to study molecular mechanisms of skin renewal and pathogenesis of dermal diseases. Horses often suffer from skin diseases, skin trauma and problems with proper wound healing, which could be improved by in vitro grown keratinocyte grafts. Herein we describe establishment and characterization of equine skin-derived primary cell cultures, using enzymatic and explant methods. The established cell lines of primary equine keratinocytes (peK) maintained high proliferative capacity for over five passages and expressed different epithelial/keratinocyte-s...
Isolation of Neutrophils from Nonhuman Species. The development of new advances in understanding the role of neutrophils in inflammation requires effective procedures for isolating and purifying neutrophils. Methods for isolating human neutrophils are fairly standard, and some are covered in other chapters of this volume and previous editions. However, procedures for isolating neutrophils from nonhuman species used to model human diseases vary from those used in isolating human neutrophils and are not as well developed. Since neutrophils are highly reactive and sensitive to small perturbations, the methods of isolation are important to avo...
Clock Genes Expression in Peripheral Leukocytes and Plasma Melatonin Daily Rhythm in Horses. In mammals, behavioral and physiological processes display 24-hour rhythms that are regulated by the circadian system. In the present study, we investigated clock gene expression in peripheral leukocytes in horses. For this purpose, 10 Italian Saddle gelding horses (9-11 years old; 475 ± 28 Kg) were housed in individual boxes under natural photoperiod and natural environmental temperature. Blood samples were collected at 4-hour intervals over a 48-hour period. The day before the start of sampling, left jugular furrow of each horse was cannulated for the blood sample collection performed in ...
Morphology, developmental stages and quality parameters of in vitro-produced equine embryos. Intracytoplasmic sperm injection (ICSI) is used to produce equine embryos invitro. The speed of embryo development invitro is roughly equivalent to what has been described for embryos produced invivo. Morphological evaluations of ICSI-produced embryos are complicated by the presence of debris and the dark nature of equine embryo cytoplasm. Morulas and early blastocysts produced invitro appear similar to those produced invivo. However, with expansion of the blastocyst, distinct differences are observed compared with uterine embryos. In culture, embryos do not undergo full expansion and thinning...
Primary Hyperparathyroidism Associated With Atypical Headshaking Behavior in a Warmblood Gelding. A 14-year-old Zweibrücker Warmblood gelding was presented for evaluation of lethargy and headshaking. The horse had a history of bouts of lameness in different limbs and back problems. It also had many mild colic episodes in the past. Results of repeat laboratory tests had shown persistent hypercalcemia (4.8 mmol/L; reference interval [RI]: 2.0-3.2 mmol/L) for 1.5 years and later on hypophosphatemia (0.4 mmol/L; RI: 0.5-1.3 mmol/L) and mild hypermagnesemia (1.0 mmol/L; RI: 0.5-0.9 mmol/L). Parathyroid hormone (PTH) concentration was within the RI. Other causes of hypercalcemia, such as...
Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection. Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22-27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at ...
Ceruloplasmin, serotransferrin and albumin presented different abundance in mares’ uterine fluid five days after insemination. Characterization of the uterine proteome before the entry of the conceptus to the uterus is essential to know the factors involved in the physiological events of gestation. The objective of the study was to compare proteomic profile of uterine fluid collected on day 5 post ovulation of cyclic and inseminated mares. Samples of endometrial secretion were recovered over 2 cycles during the fifth day post ovulation. The first cycle constituted the Cyclic group and in the following cycle, the same mares were inseminated and considered as the Inseminated group. All the samples were subjected to two-...
Could a Compliant Foam Anvil Characterize the Biofidelic Impact Response of Equestrian Helmets? The performance of equestrian helmets to protect against brain injuries caused by fall impacts against compliant surfaces such as turf has not been studied widely. We characterize the kinematic response of simulated fall impacts to turf through field tests on horse racetracks and laboratory experiments. The kinematic response characteristics and ground stiffness at different going ratings (GRs) (standard measurement of racetrack condition) were obtained from 1 m and 2 m drop tests of an instrumented hemispherical impactor onto a turf racetrack. The "Hard" rating resulted in higher peak lin...
Development of membrane-based flow-through assay for detection of trypanosomosis in equines. A rapid flow-through immunoassay using protein A labeled gold nanoparticles (GNPs) for the qualitative detection of trypanosomosis in equine serum samples was developed. The flow-through device consisted of antigen-coated nitrocellulose membrane fixed on absorbent pads. The GNPs based detection probe was used as the colored marker. The assay can be employed for the detection of specific antibodies at 14 days post-infection in the infected pooled serum samples of equines. The assay can be conducted in < 5 min. The results of the flow-through assay were comparable with the methods like E...
Establishment and application of a 10-plex liquid bead array for the simultaneous rapid detection of animal species. Meat fraud and adulteration incidents occur frequently in almost all regions of the globe, especially with the increase in the world's population. To ensure the authenticity of meat products, we developed a 10-plex xMAP assay to simultaneously detect ten animal materials: bovine, caprine, poultry, swine, donkey, deer, horse, dog, fox and mink. Results: This method was investigated by analyzing DNA extracts from raw muscle, muscle mixtures, meat products and animal feeds. Our results indicated that the species of interest can be identified, differentiated and detected down to 1 g kg in bina...
Serum protein concentrations and protein fractions in clinically healthy Italian Heavy Draft Horses using agarose gel electrophoresis. Serum protein electrophoresis (SPE) reference intervals (RIs) have been evaluated in different horses, but no specific values are shown for equine breeds as previously described in other species (dogs, cats), and no studies have been performed on SPE in draft horses. Objective: This study aimed to determine RIs for SPE in heavy draft horses (Italian Heavy Draft Horse-IHDH) living in central Italy. A comparison between different physiologic states (pregnancy and no pregnancy) and ages (foals and adults) was executed. Methods: Blood samples were collected from 215 apparently healthy horses (mare...
Effects of media and promoters on different lipid peroxidation assays in stallion sperm. Effects of different media and promoters on lipid peroxidation (LPO) in viable stallion sperm have not been reported. Aims of this study were to determine effects of three media (INRA-96™, Equipro CoolGuard™, and Biggers, Whitten and Whittingham [BWW]), and promoters (iron sulfate-Fe; ultraviolet light-UV; or control-no exposure to promoters) on viable sperm LPO using four different flow cytometric assays (i.e., BODIPY, Liperfluo, 4-hydroxylnonenal [4HNE], malonaldehyde [MDA]). Significant media x promoter interactions were detected using the Liperfluo, 4HNE, and MDA assays (P < 0.05)...
No Evidence of Mosquito Involvement in the Transmission of Equine Hepacivirus (Flaviviridae) in an Epidemiological Survey of Austrian Horses. Prevalence studies have demonstrated a global distribution of equine hepacivirus (EqHV), a member of the family Flaviviridae. However, apart from a single case of vertical transmission, natural routes of EqHV transmission remain elusive. Many known flaviviruses are horizontally transmitted between hematophagous arthropods and vertebrate hosts. This study represents the first investigation of potential EqHV transmission by mosquitoes. More than 5000 mosquitoes were collected across Austria and analyzed for EqHV ribonucleic acid (RNA) by reverse transcription quantitative polymerase chain reacti...
CENP-A binding domains and recombination patterns in horse spermatocytes. Centromeres exert an inhibitory effect on meiotic recombination, but the possible contribution of satellite DNA to this "centromere effect" is under debate. In the horse, satellite DNA is present at all centromeres with the exception of the one from chromosome 11. This organization of centromeres allowed us to investigate the role of satellite DNA on recombination suppression in horse spermatocytes at the stage of pachytene. To this aim we analysed the distribution of the MLH1 protein, marker of recombination foci, relative to CENP-A, marker of centromeric function. We demonstrated that the sa...
Evaluation of allogeneic freeze-dried platelet lysate in cartilage exposed to interleukin 1-β in vitro. Platelet-rich plasma (PRP) as well as other platelet-derived products have been used as a potential disease-modifying treatment for musculoskeletal diseases, such as osteoarthritis (OA). The restorative properties of such products rely mainly on the high concentrations of growth factors, demonstrating encouraging results experimentally and clinically. Yet, the autologous blood-derived nature of the PRP product lead to limitations that precludes it's widespread use. The main limitations for PRP use are; product variability, the need for minimum laboratory settings in most cases, and the need fo...
Preservation of fecal glucocorticoid metabolites and immunoglobulin A through silica gel drying for field studies in horses. Non-invasive methods enable stress evaluation through measuring fecal glucocorticoid metabolites (FGMs), and immunoglobulin A (IgA) in the feces avoiding stressful blood drawing or stressful restraining of animals in the field. However, FGMs and IgA are mostly analysed in freshly frozen samples, which is difficult when fresh samples cannot be frozen immediately or frozen samples cannot be stored or transported. Good results were also derived from air-dried fecal samples, which are hampered by unstable air humidity in the field. These difficulties may be overcome, when drying of samples could b...
Plasma adrenocorticotropic hormone concentration in horses decreases after freezing for 60 days. We investigated the stability of adrenocorticotropic hormone (ACTH) in plasma after freezing for different lengths of time. The plasma ACTH concentrations of 12 horses were measured on day 0 (baseline) and over time, after stimulation with thyrotropin-releasing hormone. Samples were stored at -80°C for 3, 7, 30, 60, and 90 d, or at -20°C for 3, 7, 30, and 60 d, or between ice packs at -20°C for 3 and 7 d prior to determination of ACTH concentration. ACTH concentrations were compared to baseline (non-frozen day 0 plasma) for each storage method using a mixed model with repeated measure...
Comparison of sperm selection techniques in donkeys: motile subpopulations from a practical point of view. The aim of this study was to compare the post-thaw distribution of motile sperm subpopulations, following simple or colloid centrifugation. A new analysis was used to evaluate the available number of sperm from each subpopulation after each centrifugation protocol. Frozen/thawed semen samples were divided into the following after-thawing treatments: uncentrifuged control (UDC), sperm washing (SW) and two colloid centrifugation procedures (Equipure, SLC-E, and Androcoll, SLC-A). Percentage of total and progressive motility (TM and PM), as well as sperm motility kinematics, distribution of motil...
Equine non-invasive time-lapse imaging and blastocyst development. In this study we examined the timeline of mitotic events of invitro-produced equine embryos that progressed to blastocyst stage using non-invasive time-lapse microscopy (TLM). Intracytoplasmic sperm injection (ICSI) embryos were cultured using a self-contained imaging incubator system (Miri®TL; Esco Technologies) that captured brightfield images at 5-min intervals that were then generated into video for retrospective analysis. For all embryos that progressed to the blastocyst stage, the initial event of extrusion of acellular debris preceded all first cleavages and occurred at mean (±s.e.m.)...
