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.
Collicutt NB, Garner B, Berghaus RD, Camus MS, Hart K.Although delays between blood sample collection and analysis are common in veterinary medicine, the effect of prolonged serum-clot contact time on serum glucose concentration is not well established and species differences have not been elucidated. Objective: The objective was to investigate the effect of storage time and temperature on serum glucose concentration in stored whole blood samples from horse, dog, alpaca, and sturgeon. Methods: Whole blood specimens were divided into 7 no-additive tubes and serum was separated from one sample within one hour, serving as the reference sample. The r...
Van Der Vekens N, Decloedt A, Sys S, Ven S, De Clercq D, van Loon G.Cardiac troponin I (cTnI) is a marker for detection of myocardial damage in horses. Many cTnI assays exist and medical studies have shown that the clinical performance of assays differs. The aim of this study was to compare two different cTnI assays in horses. Serum samples were taken from 23 healthy horses (group 1) and 72 horses with cardiac disease (group 2). Cardiac troponin I was determined using assay 1 in laboratory A (limit of detection, LOD, 0.03 ng/mL) and assay 2 in laboratories B and C (LOD 0.01 ng/mL). In group 1, a median cTnI concentration of <0.03 (<0.03-0.04) ng/mL and <0....
Janda J, Lehmann M, Luttmann W, Marti E.Interleukin-3 is a growth and differentiation factor for various hematopoietic cells. IL-3 also enhances stimulus-dependent release of mediators and cytokine production by mature basophils. Function of IL-3 has not been studied in horses because of lack of horse-specific reagents. Our aim was to produce recombinant equine IL-3 and test its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes (PBL). Equine IL-3 was cloned, expressed in E. coli and purified. PBL of 19 healthy and 20 insect bite hypersensitivity (IBH)-affected horses were stimulated with Culi...
Van Driessche K, Ducatelle R, Chiers K, Van Coster R, van der Kolk JH.Very few mitochondrial myopathies have been described in horses. Objective: To examine the ultrastructure of muscle mitochondria in equine cases of myopathy of unknown origin. Methods: Biopsies of vastus lateralis of the Musculus quadriceps femoris were taken predominantly immediately post mortem and processed for transmission electron microscopy. As a result, electron micrographs of 90 horses in total were available for analysis comprising 4 control horses, 16 horses suffering from myopathy and 70 otherwise diseased horses. Results: Following a thorough clinical and laboratory work-up, four o...
Sieme H, Oldenhof H.In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation secures future reproduction, and insemination doses can be easily shipped. Processing of semen for cryopreservation can be done with minimal efforts and relatively low costs. In this chapter we describe the entire cryopreservation process for stallion and bull sperm including dilution of sperm in primary and freezing extender, cooling and packaging in straws, freezing in liquid nitrogen vapor, cryogenic storage, and thawing. Special emphasis is given on preparation of commonl...
Bohórquez GA, Luzón M, Martín-Hernández R, Meana A.Although several techniques exist for the detection of equine tapeworms in serum and feces, the differential diagnosis of tapeworm infection is usually based on postmortem findings and the morphological identification of eggs in feces. In this study, a multiplex polymerase chain reaction (PCR)-based method for the simultaneuos detection of Anoplocephala magna, Anoplocephala perfoliata and Anoplocephaloides mamillana has been developed and validated. The method simultaneously amplifies hypervariable SSUrRNA gene regions in the three tapeworm species in a single reaction using three pairs of pri...
Hughes K, Scase TJ, Foote AK.Equine mammary tumors are uncommon, and relatively sparse histopathologic and molecular data exist. The present study describes the histopathologic features of 7 such tumors, which exhibited infiltrative growth, intermediate to high mitotic rates, and focally extensive necrosis. The tumors exhibited variably strong staining for vimentin and cytokeratin 14, as well as frequently weak cytoplasmic staining for pan-cytokeratin. E-cadherin expression was strong. Interestingly, a subgroup of the tumors exhibited strong nuclear staining for estrogen receptor α. Three of 7 tumors exhibited nuclear ex...
López C, Alvarez ME, Carmona JU.The aims were (1) to evaluate the bacteriostatic effect of platelet-rich plasma (PRP), platelet-rich gel (PRG), leukocyte-poor plasma (LPP), leukocyte-poor gel (LPG), plasma, and heat-inactivated plasma (IP) on both methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) over a period of 24 h; (2) to determine and to compare the concentrations and degradation over time of platelet factor 4 (PF-4), transforming growth factor beta 1 (TGF-β 1), and platelet-derived growth factor isoform BB (PDGF-BB); and (3) to identify any correlations between...
Heras S, Smits K, Leemans B, Van Soom A.Hoechst staining has traditionally been used to evaluate fertilization and parental origin of pronuclei. However, prevalence of parthenogenetic activation cannot be distinguished accurately by this protocol, and variation of relative pronuclear size and position makes it impossible to determine parental origin. We demonstrate that in equine zygotes, the epigenetic modification histone 3 lysine 9 trimethylation (H3K9me3) shows an asymmetric pattern between maternal and paternal pronuclei. H3K9me3 immunostaining appears to be a robust technique to identify the parent of origin of equine pronucle...
Dettwiler R, Schmitz AL, Plattet P, Zielinski J, Mevissen M.The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion...
Hayden SS, Blanchard TL, Brinsko SP, Varner DD, Hinrichs K, Love CC.Dilution of semen to less than 20 × 10(6) sperm/mL has been reported to decrease sperm quality in multiple species, a phenomenon known as the semen "dilution effect." Critical evaluation of stallion semen diluted to these concentrations, however, has not been reported. This study evaluated sperm motion characteristics (percent total motility [TMOT], percent progressive motility [PMOT], curvilinear velocity [μm/s], and percent straightness) and plasma membrane integrity (percent plasma membrane intact [PMI]) in semen samples diluted to 2.5 × 10(6) sperm/mL with the addition of 0%, 7.5%, ...
Heutelbeck A, Oldenhof H, Rohn K, Martinsson G, Morrell JM, Sieme H.Equipment for cryopreservation of stallion sperm is not always available. In such cases, diluted semen can be shipped to a facility for later cryopreservation. The aim of this study was to evaluate if selection of sperm via density centrifugation yields higher survival rates when cryopreservation is to be delayed (i.e. carried out after 1 day of storage at 5°C). Two-layer iodixanol as well as single-layer Androcoll density centrifugation were tested and compared with samples prepared with standard centrifugation. Special emphasis was placed on comparing centrifugation on the day of semen coll...
De Keyser K, Berth M, Christensen N, Willaert S, Janssens S, Ducatelle R, Goddeeris BM, De Cock HE, Buys N.Diagnosis of chronic progressive lymphoedema (CPL) in draught horses, including the Belgian Draught Horse, is mainly based on clinical evaluation of typical lower limb lesions. A deficient perilymphatic elastic support, caused by a pathological elastin degradation in skin and subcutis, has been suggested as a contributing factor for CPL. Elastin degradation products induce the generation of anti-elastin Ab (AEAb), detectable in horse serum by ELISA. For a clinically healthy group of draught horses, a significantly lower average AEAb-level than 3 clinically affected groups (mild, moderate and s...
Passamonti F, Vardi DM, Stefanetti V, Marenzoni ML, Prato S, Cévese P, Coletti M, Pepe M, Casagrande Proietti P, Olea-Popelka F.Early diagnosis and prevention of Rhodococcus equi pneumonia in foals represent important goals for equine clinicians. Recent protocols for diagnosis and treatment of Rhodococcosis in foals typically rely on a multimodal approach based on sonographic evidence suggestive of pyogranulomas, sonographic abscess scores and laboratory findings including plasma fibrinogen concentrations, blood biochemistry testing and platelet and leukocyte counts. The aim of this study was to assess the utility of weekly testing of serum amyloid A (SAA) and plasma fibrinogen concentrations in foals to achieve early ...
Müller C, Heidenreich E, Franzreb M, Frankenfeld K.Current purification of the glycoprotein equine chorionic gonadotropin (eCG) from horse serum includes consecutive precipitation steps beginning with metaphosphoric acid pH fractionation, two ethanol precipitation steps, and dialysis followed by a numerous of fixed-bed chromatography steps up to the specific activity required. A promising procedure for a more economic purification procedure represents a simplified precipitation process requiring only onethird of the solvent, followed by the usage of magnetic ion exchange adsorbents employed together with a newly designed 'rotor-stator' type Hi...
Córdova A, Strobel P, Vallejo A, Valenzuela P, Ulloa O, Burgos RA, Menarim B, Rodríguez-Gil JE, Ratto M, Ramírez-Reveco A.This study evaluated the effect of the use of hypometabolic TRIS extenders in the presence or the absence of AMPK activators as well as the utilization of high cooling rates in the refrigeration step on the freezability of stallion sperm. Twelve ejaculates were cryopreserved using Botucrio® as a control extender and a basic TRIS extender (HM-0) separately supplemented with 10 mM metformin, 2mM 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), 2 mM Adenosine monophosphate (AMP), 40 μM compound C AMPK inhibitor or 2 mM AMP+40 μM compound C. Our results showed that the utilization ...
Ekstrand C, Bondesson U, Gabrielsson J, Hedeland M, Kallings P, Olsén L, Ingvast-Larsson C.Detection times and screening limits (SL) are methods used to ensure that the performance of horses in equestrian sports is not altered by drugs. Drug concentration-response relationship and knowledge of concentration-time profiles in both plasma and urine are required. In this study, dexamethasone plasma and urine concentration-time profiles were investigated. Endogenous hydrocortisone plasma concentrations and their relationship to dexamethasone plasma concentrations were also explored. A single dose of dexamethasone-21-isonicotinate suspension (0.03 mg/kg) was administered intramuscularly t...
Sabban S, Ye H, Helm B.The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses ...
Machtinger ET, Geden CJ, Hogsette JA, Leppla NC.House flies, Musca domestica L., and stable flies, Stomoxys calcitrans (L.), (Diptera: Muscidae), common pests on equine facilities, were studied in the laboratory to determine the success and duration of larval development and oviposition preferences on six substrates commonly found on equine facilities. Substrates tested were hay soiled with urine and manure, fresh horse manure, pine shaving bedding soiled with urine and manure (72 h in a manure pile), builders sand bedding soiled with urine and manure aged 3 d, and soil from an overgrazed pasture mixed with urine and manure of variable age....
Meucci V, Minunni M, Vanni M, Sgorbini M, Corazza M, Intorre L.Detection of nonsteroidal anti-inflammatory drugs (NSAIDs) in equine plasma is a significant analytical problem in veterinary anti-doping controls. Results: A new HPLC method coupled to selective extraction with molecularly imprinted polymers was developed for the simultaneous determination in equine plasma of the NSAIDs phenylbutazone, flunixin, oxyphenbutazone, ketoprofen and naproxen. The analytical performances of the method have been evaluated both in standard solutions and equine plasma samples. Recovery: Molecularly imprinted polymers solid-phase extraction for all NSAIDs was >94% with ...
Hans A, Gaudaire D, Manuguerra JC, Leon A, Gessain A, Laugier C, Berthet N, Zientara S.This study shows that an unbiased amplification method applied to equine arteritis virus RNA significantly improves the sensitivity of the real-time reverse transcription-quantitative PCR (RT-qPCR) recommended by the World Organization for Animal Health. Twelve viral RNAs amplified using this method were hybridized on a high-density resequencing microarray for effective viral characterization.
Sayasith K, Sirois J, Lussier JG.The luteinizing hormone preovulatory surge stimulates several signal pathways essential for ovulation, and the regulator of G-protein signaling protein-2 (RGS2) is thought to be involved in this process. The objectives of this study were to characterize the regulation of RGS2 transcripts in equine and bovine follicles prior to ovulation and to determine its transcriptional control in bovine granulosa cells. To assess the regulation of equine RGS2 prior to ovulation, RT-PCR was performed using total RNA extracted from equine follicles collected at various times after human chorionic gonadotropi...
Nergadze SG, Belloni E, Piras FM, Khoriauli L, Mazzagatti A, Vella F, Bensi M, Vitelli V, Giulotto E, Raimondi E.Centromeres are the sites of kinetochore assembly and spindle fiber attachment and consist of protein-DNA complexes in which the DNA component is typically characterized by the presence of extended arrays of tandem repeats called satellite DNA. Here, we describe the isolation and characterization of a 137-bp-long new satellite DNA sequence from the horse genome (EC137), which is also present, even if less abundant, in the domestic donkey, the Grevy's zebra and the Burchelli's zebra. We investigated the chromosomal distribution of the EC137 sequence in these 4 species. Moreover, we analyzed its...
O'Shea CM, Werre SR, Dahlgren LA.(1) To compare the performance of 4 platelet counting technologies in equine platelet concentrates and (2) to evaluate the ability of the Magellan platelet rich plasma (PRP) system to concentrate equine platelets. Methods: Experimental study to assess method agreement. Methods: Adult mixed breed horses (n = 32). Methods: Acid citrate dextrose-A anti-coagulated whole blood was collected and PRP produced using the Magellan system according to the manufacturer's instructions. Platelets were quantified using 4 counting methods: optical scatter (Advia 2120), impedance (CellDyn 3700), hand counting,...
Lange-Consiglio A, Corradetti B, Perrini C, Bizzaro D, Cremonesi F.In human and swine, leptin (OB) has been identified in seminal plasma and leptin receptors (OB-R) on the cell surface of spermatozoa, indicating that spermatozoa are a target for OB. This hormone has also been detected in follicular fluid (FF) in women and mares, although its role requires further study. The aims of this study were to investigate the immunolocalisation and the expression of OB and OB-R in equine spermatozoa and to evaluate the involvement of OB in equine in vitro fertilisation (IVF). Since progesterone (P) and OB are both found in FF, the individual and combined effects of the...
Herrera C, Morikawa MI, Castex CB, Pinto MR, Ortega N, Fanti T, Garaguso R, Franco MJ, Castañares M, Castañeira C, Losinno L, Miragaya MH, Mutto AA.Normal mammalian early embryonic development involves apoptosis of blastomeres as a remodeling process during differentiation, starting at the blastocyst stage. Genomic DNA has been recently detected in the blastocele fluid of human embryos and has been amplified by real-time polymerase chain reaction (PCR) to diagnose the sex of in vitro-produced human embryos. This new approach varies from conventional preimplantation genetic diagnosis in that no cells are extracted from the embryo and only the blastocele fluid is aspirated and used as a DNA sample for diagnosis. In the present work, we inv...
Yoon M, Jiang J, Chung KH, Roser JF.Insulin-like growth factor plays a paracrine/autocrine role in regulating testicular function in the stallion, but its presence in the equine epididymis remains unknown. The aim of this study was to test the hypothesis that insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR) are localized in the caput, corpus, and cauda of the epididymis in an age-dependent manner. Immediately after castration, epididymal tissue was fixed, paraffin-embedded, and processed for immunohistochemistry (IHC). Western blot was also performed using equine epididymal extracts to verify the specificity of t...
Sykora S, Brandt S.Equine hoof canker is a chronic pododermatitis of still unknown aetiology. Recent findings reported for 3 canker-bearing individuals are suggestive for Treponema spp. having a role in disease pathogenesis. Objective: Based on this hypothesised association, we assessed a larger number of DNA samples from hooves with canker and normal hooves for the presence of treponemal DNA. Methods: Retrospective survey of archived material. Methods: The study involved 71 archival, PCR-compatible DNA extractions purified from 59 canker samples obtained from 26 equine cases and from 12 hoof biopsies taken from...
Scopel e Silva D, de Castro CC, da Silva e Silva F, Sant'anna V, Vargas GD, de Lima M, Fischer G, Brandelli A, da Motta Ade S, Hübner Sde O.P34 is an antimicrobial peptide produced by a Bacillus sp. strain isolated from the intestinal contents of a fish in the Brazilian Amazon basin with reported antibacterial activity. The aim of this work was to evaluate the peptide P34 for its in vitro antiviral properties against canine adenovirus type 2 (CAV-2), canine coronavirus (CCoV), canine distemper virus (CDV), canine parvovirus type 2 (CPV-2), equine arteritis virus (EAV), equine influenza virus (EIV), feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1). The results showed that the peptide P34 exhibited antiviral activity a...
Tydén E, Tjälve H, Larsson P.Among the cytochrome P450 enzymes (CYP), families 1-3 constitute almost half of total CYPs in mammals and play a central role in metabolism of a wide range of pharmaceuticals. This study investigated gene and protein expression and cellular localisation of CYP1A, CYP2A, CYP2C, CYP2D and CYP2E in equine intestine and liver. Real-time polymerase chain reaction (RT-PCR) was used to analyse gene expression, western blot to examine protein expression and immunohistochemical analyses to investigate cellular localisation. Results: CYP1A and CYP2C were the CYPs with the highest gene expression in the ...
Nemoto M, Ohta M, Tsujimura K, Bannai H, Yamanaka T, Kondo T, Matsumura T.We evaluated loop-mediated isothermal amplification (LAMP) as a means of detecting equine herpesvirus type 1 (EHV-1) DNA directly from nasal swabs. To increase the sensitivity, we added a step in which the samples were heat-treated to the original LAMP procedure. The detection limit of the LAMP assay with heat treatment was 10 times more sensitive than the original LAMP assay even when the DNA extraction step was omitted. In addition, the LAMP assay with heat treatment was more sensitive than the original LAMP assay and the polymerase chain reaction using clinical samples. The LAMP assay with ...
Cook RF, Sinclair R, Mumford JA.An antigen capture indirect enzyme linked immunosorbent assay (ELISA) was developed to detect influenza nucleoprotein antigen in nasal secretions from horses infected with A/equine/H3N8 viruses. Results from this assay were compared with conventional virus isolation in embryonated hens eggs.
Hosaka Y, Teraoka H, Yamamoto E, Ueda H, Takehana K.The aim of the present study was to clarify the presence and determine the role of apoptosis in the degenerative process of the superficial digital flexor tendon (SDFT) in the horse. Samples were obtained from normal and inflamed SDFTs of horses. To detect apoptosis and to identify apoptotic cells, the samples were subjected to immunohistochemical labelling and Western blot analysis. Although a large number of cells in degenerate areas showed positive reactions with caspase-3 and single stranded DNA antibodies, cells in normal tendon samples showed very weak reactions. Excessive apoptosis was ...
Amavisit P, Browning GF, Lightfoot D, Church S, Anderson GA, Whithear KG, Markham PF.A rapid polymerase chain reaction (PCR) assay was developed for detecting Salmonella in faeces of horses and assessed on samples from horses admitted to a veterinary hospital. Direct detection was achieved by amplification of part of ompC after extraction of DNA from faeces using a spin column method to reduce the amount of inhibitory substances in samples. An internal positive control was included to detect false negative results. While the sensitivity of the PCR assay was less than culture when assessed on faeces inoculated with Salmonella, its sensitivity on faecal samples obtained from hor...
Carvalho GM, Ramos CP, Lobato FCF, Guedes RMC, Giaretta PR, Silva ROS.Despite the known importance of Clostridioides (Clostridium) difficile infection (CDI) in animals, there are no published guidelines for the diagnosis of CDI. The performance of the available commercial methods, all standardized for human stool samples, can vary according to the animal species. Thus, the aim of the present study was to review the literature on the detection of C. difficile in pigs, horses, and dogs. The detection of toxins A and B using enzyme immunoassays seems to have low performance in piglet and dog samples, while it shows high sensitivity for the diagnosis of CDI in foal...
Badr Y, Okada A, Abo-Sakaya R, Beshir E, Ohya K, Fukushi H.Equine herpesvirus type 1 (EHV-1) UL11 is a 74-amino-acid tegument protein encoded by ORF51 of the EHV-1 genome. EHV-1 UL11 was previously reported by other researchers using the RacL22 and RacH strains to be nonessential for viral replication in cultured cells. Here, we constructed UL11 mutant viruses including a UL11 null mutant and three C-terminal truncated mutants, for further characterization of EHV-1 UL11 using bacterial artificial chromosome (BAC) technology based on the neuropathogenic strain Ab4p. EHV-1 Ab4p UL11 was localized to juxtanuclear and Golgi regions as reported by other re...
Nishita T, Matsushita H.1. Carbonic anhydrase III (CA-III) from different mammalian species (horse, cow, dog, cat, rat and rabbit) has been analyzed by the immunodiffusion technique with anti-equine CA-III serum. 2. Immunodiffusion demonstrated the absence of cross-reactivity between isozyme CA-I, CA-II, and CA-III. 3. Cross-reactions were observed between the CA-III from all the species examined except the rabbit. 4. Molecular weights and isoelectric points of CA-III from different species were determined by Western blotting.
Legacki EL, Scholtz EL, Ball BA, Esteller-Vico A, Stanley SD, Conley AJ.Few studies have provided a longitudinal analysis of systemic concentrations of conjugated oestrogens (and androgens) throughout pregnancy in mares, and those only using immunoassay. The use of liquid chromatography tandem mass spectrometry (LC-MS/MS) will provide more accurate concentrations of circulating conjugated steroids. Objective: To characterise circulating concentrations of individual conjugated steroids throughout equine gestation by using LC-MS/MS. Methods: Longitudinal study and comparison of pregnant mares treated with vehicle or letrozole in late gestation. Methods: Sulphated oe...
Massányi P, Trandzik J, Nad P, Toman R, Skalická M, Koréneková B.To determine the seminal concentrations of copper, zinc, iron, cadmium, lead and nickel in bulls, rams, boars, stallions and foxes and study their correlations. Methods: Semen samples were obtained, digested and analyzed by means of the atomic absorption spectrophotometer. Data were analyzed statistically with the Student's t-test and Scheffe's test using PC programs SAS and Excel. Results: The seminal copper concentration was significantly higher in ram [(2.49+/-0.18) mg/kg] and fox [(2.16+/-0.53) mg/kg] than that in bull [(1.64+/-0.21) mg/kg], boar [(1.64+/-0.28) mg/kg] and stallion (0.86 mg...
Ward TL, Valberg SJ, Lear TL, Guérin G, Milenkovic D, Swinburne JE, Binns MM, Raudsepp T, Skow L, Chowdhary BP, Mickelson JR.Comparative biochemical and histopathological data suggest that a deficiency in the glycogen branching enzyme (GBE) is responsible for a fatal neonatal disease in Quarter Horse foals that closely resembles human glycogen storage disease type IV (GSD IV). Identification of DNA markers closely linked to the equine GBE1 gene would assist us in determining whether a mutation in this gene leads to the GSD IV-like condition. FISH using BAC clones as probes assigned the equine GBE1 gene to a marker deficient region of ECA26q12-->q13. Four other genes, ROBO2, ROBO1, POU1F1, and HTR1F, that flank GB...
Cawley A, Keen B, Tou K, Elbourne M, Keledjian J.The concept of biomarker measurements in the form of a ratio has not been explored in detail. This is surprising considering the current and future potential for biomarkers incorporating endogenous reference compounds (ERCs) in a range of fields. A selection of these relating to clinical and forensic applications, human antidoping, equine antidoping and veterinary residues are discussed.
Valberg SJ, Soave K, Williams ZJ, Perumbakkam S, Schott M, Finno CJ, Petersen JL, Fenger C, Autry JM, Thomas DD.Sarcolipin (SLN), myoregulin (MRLN), and dwarf open reading frame (DWORF) are transmembrane regulators of the sarcoplasmic reticulum calcium transporting ATPase (SERCA) that we hypothesized played a role in recurrent exertional rhabdomyolysis (RER). Objective: Compare coding sequences of SLN, MRLN, DWORF across species and between RER and control horses. Compare expression of muscle Ca2+ regulatory genes between RER and control horses. Methods: Twenty Thoroughbreds (TB), 5 Standardbreds (STD), 6 Quarter Horses (QH) with RER and 39 breed-matched controls. Methods: Sanger sequencing of SERCA reg...
Chowdhary BP, Harbitz I, Davies W, Gustavsson I.In situ hybridization techniques were used to localize regionally the calcium release channel (CRC) gene on cattle and horse chromosomes, using a porcine CRC cDNA probe. In cattle, the hybridization signal peaked on the 18q23-q26 bands and in horse on the 10pter region. Previous studies have shown that the glucose phosphate isomerase (GPI) gene localizes at the same site in both species, indicating that the two loci are syntenic. As CRC and GPI are syntenic in human, pig and mouse, the present results in cattle and horse represent another example of synteny conservation in the evolution of mam...
Williams SM, Fulton RM, Patterson JS, Reed WM.The diagnosis of eastern equine encephalitis (EEE) virus infection in avian species is relatively difficult when compared with other species. There are no characteristic histologic lesions in the avian brain that would serve to distinguish EEE from infections with, for example, Newcastle disease or highly pathogenic avian influenza virus. Traditionally, virus isolation (VI) and/or hemagglutination inhibition (HI) has been used for a definitive diagnosis of EEE in birds. Recently, we developed an immunohistochemistry (IHC) technique for confirmatory diagnosis of EEE infection in equine brain. T...
Blähser S.In the thyroid glands of the horse, pig, deer, mole, and rat, C-cells could be demonstrated by means of the immunocytochemical PAP-technique using rabbit antisera against human calcitonin. Only in ruminants, the cross-reaction between the intracellularly stored antigen and the antibodies used appeared to be incomplete.
Karasek M, Marek K, Kunert-Radek J.The influence of gonadotropic hormones on the ultrastructure of rat pinealocytesin short-term organ culture was studied. Human chorionic gonadotropin (HCG), as well as pregnant mare serum gonadotropin (PMSG), caused a marked activation of pinealocytes. An hypothesis is discussed implying the presence of a feedback mechanism between the pineal organ and the hypothalamo-hypophysial system.
Khan I, Wieler LH, Saqib M, Melzer F, Santana VL, Neubauer H, Elschner MC.The complement fixation test (CFT) is the only serological test prescribed by the World Organisation for Animal Health (OIE) for the diagnosis of glanders in international trading of equids. However, false-positive reactions have caused financial losses to the animal owners in the past, and false-negative tests have resulted in the introduction of glanders into healthy equine populations in previously glanders-free areas. Both warm (incubation at 37°C for 1 h) and cold (overnight incubation at 4°C) procedures are recommended by the OIE for serodiagnosis of glanders. In a comparison of the se...
Harrington JR, Golding MC, Martens RJ, Halbert ND, Cohen ND.To evaluate a real-time quantitative polymerase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi. Methods: 1 virulent, 2 intermediately virulent, and 2 avirulent strains of R. equi and 16 isolates of bacteria genetically related to R. equi. Methods: The QPCR assay was evaluated for detection and quantitation of the virulence-associated gene (vapA) of R. equi in pure culture and in samples of tracheobronchial fluid, which were inoculated with known numbers of virulent R. equi. Results were compared with those derived via quantitative microbial culture a...
Blong AE, Epstein KL, Brainard BM.To investigate the in vitro effects of 3 hydroxyethyl starch (HES) solutions on viscoelastic coagulation testing and platelet function in horses. Methods: Blood samples collected from 7 healthy adult horses. Methods: Blood samples were diluted with various crystalloid and HES solutions to approximate the dilution of blood in vivo that occurs with administration of a 10 and 20 mL/kg fluid bolus to a horse (1:8 and 1:4 dilutions, respectively). Diluted samples were analyzed through optical platelet aggregometry, platelet function analysis, thromboelastography, and dynamic viscoelastic coagulomet...
von Fellenberg R, Kohler L, Grünig G, Pellegrini A.Neutral neutrophil protease, elastase activities, and cytosol protease inhibitors of these enzymes of horses and man were compared. Human neutrophils had 5 times the elastase activity of equine neutrophils, and neutral protease activity was approximately 50% greater in human neutrophils than that in equine neutrophils. Cytosol inhibitors for elastase and neutral proteases were not found in human neutrophils, whereas large amounts were found in equine neutrophils. Using fibrinogen-agarose electrophoresis, 4 cytosol inhibitors of different enzyme specificities were detected. These cytosol inhibi...
Morita H, Shimazu M, Shiono H, Toh H, Nakajima F, Akita H, Takagi M, Takami H, Murakami M, Masaoka T, Tanabe S, Hattori M.We previously isolated five strains of putative lactobacilli from the faeces of a thoroughbred horse (a 4-year-old male). Of the five strains, four were identified as members of existing Lactobacillus species; however, sequence analysis of the 16S rRNA gene revealed that the fifth isolate, DI70(T), showed approximately 97 % identity (1325/1366 bp) with the type strain of Lactobacillus delbrueckii. Therefore, we considered the possibility that DI70(T) represents a novel species of the genus Lactobacillus. Cells of strain DI70(T) were Gram-stain-positive, catalase-negative, non-spore-forming, no...
Daneshvar Amoli A, Mohebali N, Farzaneh P, Shahzadeh Fazeli SA, Nikfarjam L, Ashouri Movasagh S, Moradmand Z, Ganjibakhsh M, Nasimian A, Izadpanah M....Caspian horse, a rare horse breed found in 1965 by Louise Firouz in northern Iran, is a small horse which is reported to be in danger of extinction in its original homeland. There seems to be a great need to prevent extinction of this valuable horse. In this study, 51 fibroblast cell lines from Caspian horse ear marginal tissue were successfully established by sampling 60 horses using primary explant technique. Cells were authenticated and growth curve was plotted. According to results obtained, population doubling time (PDT) was calculated 23 ± 0.5 h for all cell lines. Multiplex polymera...
Leemans B, Gadella BM, Stout TA, Sostaric E, De Schauwer C, Nelis H, Hoogewijs M, Van Soom A.In many species, sperm binding to oviduct epithelium is believed to be an essential step in generating a highly fertile capacitated sperm population primed for fertilization. In several mammalian species, this interaction is based on carbohydrate-lectin recognition. D-galactose has previously been characterized as a key molecule that facilitates sperm-oviduct binding in the horse. We used oviduct explant and oviduct apical plasma membrane (APM) assays to investigate the effects of various carbohydrates; glycosaminoglycans; lectins; S-S reductants; and the capacitating factors albumin, Ca(2+) a...
Quiñones-Pérez C, Martínez A, Ortiz I, Crespo F, Vega-Pla JL.Despite the advances in reproductive technology, there is still a considerable number of low sperm quality cases in stallions. Recent studies in humans have detected several seminal microflora-spermatozoa associations behind some idiopathic infertility cases. However, no studies are available on horses, and there is limited information on the microflora present in stallion ejaculates. Accordingly, the objective of this study was to examine associations to the presence of bacteria families with five sperm quality parameters: concentration, total number of spermatozoa, total and progressive moti...
Jasko DJ.This article outlines a basic method for conducting a stallion semen evaluation. After the removal of the gel fraction of the ejaculate, semen gel-free volume is determined, and any abnormality in appearance is noted. Concentration of sperm cells in semen can be determined with the use of either a hemacytometer or spectrophotometer after appropriate dilution of raw semen. The percentage of progressively motile sperm is evaluated promptly after collection of semen with the use of a phase-contrast microscope. The total numbers of sperm and progressively motile sperm in the ejaculate are calculat...
Herrler A, Pell JM, Allen WR, Beier HM, Stewart F.Insulin-like growth factor-I (IGF-I) promotes early embryonic development in several species. In the rabbit, IGF-I binds to the embryonic coats from Day 3 of development onward by a 38-kDa protein that is probably insulin-like growth factor-binding protein 3 (IGFBP3). In the present study, ligand, Western, and Northern blot analyses were used to demonstrate the presence of IGF-I-binding activity, several immunoreactive IGFBP3 proteins, and IGFBP3 mRNA in horse conceptuses with particularly large amounts of immunoreactive IGFBP3 in the conceptus capsule. In addition, immunoprecipitation of radi...
House C, Mikiciuk PE, Berninger ML.Five serological methods of diagnosing African horse sickness were evaluated, using a battery of serum samples from experimental horses vaccinated and challenged with each serotype of African horse sickness virus (AHSV1 through AHSV9): agar gel immunodiffusion (AGID), indirect fluorescent antibody (IFA), complement fixation (CF), virus neutralization (VN), and enzyme-linked immunosorbent assay (ELISA). The 5 tests were also compared using a panel of field samples, convalescent equine sera with antibodies to domestic equine viral diseases, and sera from horses awaiting export. The ELISA describ...
van der Merwe D, van den Wollenberg L, van Hees-Valkenborg J, de Haan T, van der Drift S, Vandendriessche V.Analysis of hair to gain insight into the trace mineral status and exposure to toxic heavy metals of horses is attractive because hair is an easily accessible sample material. To investigate the potential value of hair analysis in horses for determination of trace mineral and heavy metal concentrations, we analyzed mane hair and liver samples from 62 horses presented for slaughter at a facility in the Netherlands that receives horses from all regions of the country. Hair samples were cleaned in warm water. After acid digestion of hair and liver specimens, we quantified, with inductively couple...
Walker JA, Timoney JF.Streptococcus equi causes equine strangles, a purulent lymphadenopathy of the head and neck. An avirulent, non-encapsulated strain (Pinnacle) has been used widely in North America as an intranasal vaccine. The aim of the study was to create a specific mutation of the hyaluronate synthase (hasA) gene in Pinnacle to permanently abolish the production of capsule and provide an easily recognisable genetic marker. An internal fragment of hasA was generated by PCR and cloned into pTW100 (Microscience, UK). An encapsulated revertant of Pinnacle was then transformed with the recombinant plasmid by ele...
Rampin A, Skoufos I, Raghunath M, Tzora A, Diakakis N, Prassinos N, Zeugolis DI.The absence of a native extracellular matrix and the use of xenogeneic sera are often associated with rapid tenocyte function losses during in vitro culture. Herein, we assessed the influence of different sera (equine serum and foetal bovine serum) on equine tenocyte morphology, viability, metabolic activity, proliferation and protein synthesis as a function of tissue-specific extracellular matrix deposition (induced via macromolecular crowding), aging (passages 3, 6, 9) and time in culture (days 3, 5, 7). In comparison to cells at passage 3, at day 3, in foetal bovine serum and without macrom...
