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.
Driessen B, Scandella M, Zarucco L.To develop a technique for placing continuous peripheral nerve block (CPNB) catheters adjacent to palmar nerves in horses and to evaluate the effect of low-volume local anesthetic (LA) infusion on nociception in the distal equine thoracic limb. Methods: In vitro and in vivo laboratory investigation. STUDY MATERIAL AND ANIMALS: Forty-two thoracic limbs from 22 equine cadavers and five horses. Methods: Thoracic limb specimens were dissected to find landmarks for catheter insertion adjacent to medial and lateral palmar nerves. Based on the anatomy of the proximal metacarpus, a technique for placi...
Nitschke M, Korte T, Tielesch C, Ter-Avetisyan G, Tünnemann G, Cardoso MC, Veit M, Herrmann A.Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. Infection by EAV requires the release of the viral genome by fusion with the respective target membrane of the host cell. We have investigated the entry pathway of EAV into Baby Hamster Kidney cells (BHK). Infection of cells assessed by the plaque reduction assay was strongly inhibited by substances which interfere with clathrin-dependent endocytosis and by lysosomotropic compounds. Furthermore, infection of BHK cells was suppressed when clathrin-dependent endocytosis was inhibited ...
Waite JA, Love CC, Brinsko SP, Teague SR, Salazar JL, Mancill SS, Varner DD.Two experiments were conducted to investigate modifications in cushioned centrifugation of stallion semen. Specifically, the effects of tube type, centrifugation medium, cushion type, and centrifugation force on post-centrifugation sperm recovery rate and quality were evaluated. In Experiment 1, sperm recovery rate was higher (P<0.05) in conventional plastic conical-bottom tubes (103%) than in newly developed glass nipple-bottom tubes (96%) following cushioned centrifugation; however, several measures of semen quality (i.e., % total motility [MOT], % progressive motility [PMOT], curvilinear...
Ellenberger C, Wilsher S, Allen WR, Hoffmann C, Kölling M, Bazer FW, Klug J, Schoon D, Schoon HA.Previous studies have shown that the equine uterus produces many progesterone-dependent proteins throughout gestation. In particular, uterocalin and uteroferrin are detectable using electrophoresis or blot analyses but information regarding the immunohistochemical placental distribution of these two proteins is rare and information regarding uteroglobin is still lacking. The aim of the present study was to co-immunolocalise these three secretory proteins in the mare's uterus throughout gestation in an effort to understand their functional role in the maintenance of pregnancy. Therefore, endome...
Bauer N, Moritz A.Besides flow cytometric detection of cellular hemoglobin (HGB) concentration, the ADVIA 2120 uses a novel cyanide-free colorimetric method to determine extracellular total HGB concentration. In human samples, the results are equivalent to those of the cyanmethemoglobin method on the ADVIA 120. Cyanide-free HGB measurement has not been evaluated in animal samples. Objective: The aim of this prospective study was to compare the 3 methods of HGB analysis on the ADVIA 2120 and ADVIA 120 in blood samples from dogs, cats, and horses. Methods: Consecutive fresh K(3)EDTA blood samples from 119 dogs, 1...
Turton JF, Baklan H, Siu LK, Kaufmann ME, Pitt TL.A multiplex PCR using targets within the serotype-specific region of the capsular polysaccharide synthesis gene cluster of serotypes K1, K2 and K5 was evaluated using the 77 reference serotype strains of Klebsiella, and a panel of clinical isolates subjected previously to conventional serotyping. The PCR was highly specific for these serotypes, which are those most associated with virulence in humans and horses. PCR confirmed that isolates of the K5 serotype had cross-reacted with antiserum for other serotypes, particularly for K7. K5 isolates received by our laboratory were almost exclusively...
Wagner B, Hillegas JM, Flaminio MJ, Wattrang E.Interferon-alpha (IFN-alpha) is a type I interferon that is secreted during the early stages of the innate immune response and is often induced upon infection with viral pathogens. IFN-alpha production affects multiple downstream events influencing both innate and adaptive immune responses. Here, we describe the expression of an equine rIFN-alpha/IgG4 fusion protein in mammalian cells. The anti-viral activity of rIFN-alpha/IgG4 was found to be 70-fold higher than that of a previously described IFN-gamma/IgG1 as tested by bioassay. The purified rIFN-alpha was subsequently used for the generatio...
Ishii M, Kobayashi S, Acosta TJ, Miki W, Yamanoi T, Matsui M, Miyake Y, Miyamoto A.The aim of this study was to clarify the relationship between circulating oxytocin (OT) and PGF(2alpha) metabolite (PGFM) in mares at the third stage of labor and placental expulsion time in order to investigate a cause of retained placenta of which the incidence increase in a heavy draft mare. Blood was sampled every 5 min from foaling to expulsion of the placenta in 18 heavy draft mares to evaluate circulating OT and PGFM. The relationships between OT and PGFM concentration and recorded placental expulsion times were investigated. The results were as follows (1) The highest level of OT conce...
Varner DD.The conventional approach to evaluation of stallion semen dates back several decades, and includes evaluation of spermatozoal concentration, semen volume, spermatozoon morphological characteristics, and spermatozoal motility patterns initially and following in-vitro storage. While an analysis performed in this manner does have predictive value, incorporation of some more newly developed techniques may improve the predictive value of the examination. This communication addresses some newer tests that can be applied today for evaluation of semen, as well some tests that may be available in the c...
Brandt S, Haralambus R, Schoster A, Kirnbauer R, Stanek C.Bovine papillomaviruses of types 1 and 2 (BPV-1 and -2) chiefly contribute to equine sarcoid pathogenesis. However, the mode of virus transmission and the presence of latent infections are largely unknown. This study established a PCR protocol allowing detection of <or=10 copies of the BPV-1/-2 genes E5 and L1. Subsequent screening of peripheral blood mononuclear cell (PBMC) DNA derived from horses with and without BPV-1/2-induced skin lesions demonstrated the exclusive presence of E5, but not L1, in PBMCs of BPV-1/2-infected equines. To validate this result, a blind PCR was performed from ...
Bai Y, Tong TG, Zhang WJ, Xu SL, Wang Q, Liu GL, Wu DL.To develop a quantitative ELISA by measuring interferon (IFN-gamma) of equine lymphocytes. Methods: Sandwich ELISA for equine IFN-gamma was developed using mAb A5 as a capture antibody and biotinylated mAb SB10 as a detection antibody. Results: The detection limit of the sandwich ELISA for equine IFN-gamma was 1 microg/L and did not show cross-reactivity with recombinant equine IL-18. Equine IFN-gamma was detected by ELISA in culture medium of the peripheral blood mononuclear cells (PBMCs) stimulated with ConA or PMA/Ionomycin. Conclusions: This method can be used to help understand the role o...
Edwards JF.A review of the pathologic conditions of the stallion reproductive tract is presented. The stallion has a number of lesions similar to those of other male domestic species but also has several unique to the horse. Some are diagnosed infrequently now because of new disease control measures and new husbandry practices. Modern immunostaining and molecular techniques should be applied to better characterize pathologic conditions in the stallion.
Agüero M, Gómez-Tejedor C, Angeles Cubillo M, Rubio C, Romero E, Jiménez-Clavero A.African horse sickness is an arthropod-borne disease of the equine included in the World Organization for Animal Health (OIE) list with important economic consequences for horse trade. The disease is caused by African horse sickness virus (AHSV; family Reoviridae, genus Orbivirus), which is transmitted by Culicoides midges. It is endemic in sub-Saharan Africa, spreading occasionally outside this area where the occurrence of Culicoides vectors allows virus transmission. Currently, only conventional (gel-based) reverse transcription polymerase chain reaction (RT-PCR) protocols are available for ...
Ho EN, Wan TS, Wong AS, Lam KK, Stewart BD.Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to detect effectively the use of insulins in horses would be required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. This study describes a method for the simultaneous detection of bovine, porcine and human...
Brinkmeyer-Langford C, Raudsepp T, Gustafson-Seabury A, Chowdhary BP.A total of 207 BAC clones containing 155 loci were isolated and arranged into a map of linearly ordered overlapping clones over the proximal part of horse chromosome 21 (ECA21), which corresponds to the proximal half of the short arm of human chromosome 19 (HSA19p) and part of HSA5. The clones form two contigs - each corresponding to the respective human chromosomes - that are estimated to be separated by a gap of approximately 200 kb. Of the 155 markers present in the two contigs, 141 (33 genes and 108 STS) were generated and mapped in this study. The BACs provide a 4-5x coverage of the regio...
Lear TL, Bailey E.Cytogenetic analyses of horses have benefited the horse industry by identifying chromosomal aberrations causing congenital abnormalities, embryonic loss and infertility. Technical advances in cytogenetics enabled the identification of chromosome specific aberrations. More recently, advances in genomic tools have been used to more precisely define chromosome abnormalities. In this report we review the history of equine clinical cytogenetics, identify historical landmarks for equine clinical cytogenetics, discuss how the current use of genomic tools has benefited this area, and how future genomi...
Quan M, van Vuuren M, Howell PG, Groenewald D, Guthrie AJ.Between 2004 and 2006, 145 African horse sickness viruses (AHSV) were isolated from blood and organ samples submitted from South Africa to the Faculty of Veterinary Science, University of Pretoria. All nine serotypes were represented, with a range of 3-60 isolates per serotype. The RNA small segment 10 (S10) nucleotide sequences of these isolates were determined and the phylogeny investigated. AHSV, bluetongue virus (BTV) and equine encephalosis virus (EEV) all formed monophyletic groups and BTV was genetically closer to AHSV than EEV. This study confirmed the presence of three distinct S10 ph...
Brandt S, Haralambus R, Shafti-Keramat S, Steinborn R, Stanek C, Kirnbauer R.Bovine papillomavirus type 1 or 2 (BPV-1, BPV-2) are accepted causal factors in equine sarcoid pathogenesis. Whereas viral genomes are consistently found and expressed within lesions, intact virions have never been detected, thus permissiveness of sarcoids for BPV-1 replication remains unclear. To reassess this issue, an immunocapture PCR (IC/PCR) was established using L1-specific antibodies to capture L1-DNA complexes followed by amplification of the viral genome. Following validation of the assay, 13 sarcoid-bearing horses were evaluated by IC/PCR. Samples were derived from 21 tumours, 4 per...
Vonaparti A, Lyris E, Panderi I, Koupparis M, Georgakopoulos C.Two simple and rapid LC/MS methods with direct injection analysis were developed and validated for the quantification and identification of hydrocortisone in equine urine using the same sample preparation but different mass spectrometric systems: ion trap mass spectrometry (IT-MS) and time-of-flight mass spectrometry (TOF-MS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as particle-free diluted urine samples were directly injected into both LC/MS systems. Desonide was used as internal standard (IS). The linear range was 0.25-2.5 microg ml(-1) for...
Westermann CM, Dorland L, Votion DM, de Sain-van der Velden MG, Wijnberg ID, Wanders RJ, Spliet WG, Testerink N, Berger R, Ruiter JP, van der Kolk JH.The aim of the current study was to assess lipid metabolism in horses with atypical myopathy. Urine samples from 10 cases were subjected to analysis of organic acids, glycine conjugates, and acylcarnitines revealing increased mean excretion of lactic acid, ethylmalonic acid, 2-methylsuccinic acid, butyrylglycine, (iso)valerylglycine, hexanoylglycine, free carnitine, C2-, C3-, C4-, C5-, C6-, C8-, C8:1-, C10:1-, and C10:2-carnitine as compared with 15 control horses (12 healthy and three with acute myopathy due to other causes). Analysis of plasma revealed similar results for these predominantly...
Endo A, Roos S, Satoh E, Morita H, Okada S.Two strains of lactic acid bacteria were isolated from faeces of two actively racing thoroughbred horses. The isolates formed a subcluster in the Lactobacillus reuteri phylogenetic group, closely related to Lactobacillus fermentum, L. gastricus, L. ingluviei and L. mucosae, by phylogenetic analysis based on 16S rRNA gene sequences. Levels of DNA-DNA relatedness revealed that the isolates belonged to the same taxon and were genetically separated from their phylogenetic relatives. Biochemical and physiological characteristics also distinguished the isolates from their phylogenetic relatives. The...
Hastie PM, Mitchell K, Murray JA.There is a need to further our understanding of the role that the equine hindgut ecosystem plays in digestive processes and diseases. The aim of the present study was to utilise the real-time PCR technique to determine the abundance of candidate cellulolytic (Ruminococcus flavefaciens; Fibrobacter succinogenes) and non-cellulolytic (Streptococcus bovis) bacteria in lumen contents from the caecum, ventral and dorsal colon, and rectum of healthy horses (n 14). Total DNA was extracted from frozen and lyophilised lumen contents, and PCR primers and Taqman probes were designed based on 16S rDNA seq...
Giordano A, Rossi G, Pieralisi C, Paltrinieri S.The ADVIA 120 is an automated laser cell counter widely used in veterinary medicine. Although specific software for equine samples is available and validated, only a few reports have been published comparing the ADVIA 120 with other methods for equine hemogram evaluation. Objective: The purpose of this study was to compare the hematologic values and reference intervals obtained on the ADVIA 120 with those obtained on an impedance cell counter and manual differential counts in healthy horses. Methods: EDTA-anticoagulated blood samples were obtained from 114 clinically healthy horses of various ...
Owens SD, Snipes J, Magdesian KG, Christopher MM.Blood typing before transfusion minimizes the risk of transfusion reactions and prevents immunization of the recipient against incompatible RBC antigens. The major RBC antigens that warrant identification before packed RBC or whole blood transfusions in horses are Ca and Aa. Standard blood-typing protocols are time-consuming (2.5-3.0 hours) and impractical in emergency settings. Objective: The purpose of this study was to determine whether equine RBCs could be typed for Ca and Aa antigens using sera from horses with RBC antibodies in a modified rapid (15 minute) blood-typing protocol. Methods:...
Colas C, Garcia P, Popot MA, Bonnaire Y, Bouchonnet S.Solid-phase extraction cartridges among those usually used for screening in horse doping analyses are tested to optimize the extraction of harpagoside (HS), harpagide (HG), and 8-para-coumaroyl harpagide (8PCHG) from plasma and urine. Extracts are analyzed by liquid chromatography coupled with multi-step tandem mass spectrometry. The extraction process retained for plasma applies BondElut PPL cartridges and provides extraction recoveries between 91% and 93%, with RSD values between 8 and 13% at 0.5 ng/mL. Two different procedures are needed to extract analytes from urine. HS and 8PCHG are extr...
Matiasek K, Gais P, Rodenacker K, Jütting U, Tanck JJ, Schmahl W.Stereological techniques have been increasingly employed for assessment and characterization of neuromuscular diseases in humans and animals. As an adjunct to histopathology, morphometrical algorithms provide quantitative evidence of the peripheral nerve composition, thereby shedding light on its fibre characteristics and basic electrophysiological properties. In the horse, stereological investigations already have focussed on the recurrent laryngeal, deep peroneal and lateral palmar nerves (LPN). Of these, only the latter is suitable for taking biopsies in clinical settings, however, it does ...
Andrews FM, Buchanan BR, Elliott SB, Al Jassim RA, McGowan CM, Saxton AM.Volatile fatty acids, byproducts of carbohydrate fermentation by resident bacteria, have been implicated in causing nonglandular (NG) gastric ulcers. Lactic acid (LA), also produced by stomach bacteria, may cause gastric ulcers when exposed to the equine NG mucosa. Objective: To investigate the in vitro effects of LA on equine NG mucosa bioelectric properties, sodium transport and tissue resistance. Methods: Gastric tissues obtained from 13 mature horses were studied in Ussing chambers. Short-circuit current (Isc) and potential difference (PD) were measured, and electrical resistance (R) and c...
Lu Z, Branscum AJ, Shuck KM, Zhang J, Dubovi EJ, Timoney PJ, Balasuriya UB.Two previously developed TaqMan fluorogenic probe-based 1-tube real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays (T1 and T2) were compared and validated for the detection of Equine arteritis virus (EAV) nucleic acid in equine semen and tissue culture fluid (TCF). The specificity and sensitivity of these 2 molecular-based assays were compared to traditional virus isolation (VI) in cell culture. The T1 real-time RT-PCR had a higher sensitivity (93.4%) than the T2 real-time RT-PCR (42.6%) for detection of EAV RNA in semen. However, the T1 real-time RT-PCR was les...
Peng Z, Shen D, Zhang D, Li X, Wang L, Zhai Q, Hou Z, Li H.The equine Parascaris spp. is large, parasitic nematodes, and predominantly focuses on the intestine of foals and young weanlings. There are two roundworms, Parascaris equorum and Parascaris univalens, recognized among equine hosts. In this study, all fifty-nine Parascaris worms were harvested from three different equine hosts (twenty specimens from Equus zebra, twenty specimens from E. caballus, and nineteen specimens from E. asinus). The ribosomal gene (ITS) and mitochondrial genes (cox1 and nadh1) were amplified to identify and genetically characterize these worms. Analysis of ITS sequences...
The measurement of cell proliferation and cell viability using 5'bromo-2'deoxy-uridine (BrdU) labelling has been described in several cell types and species. The aim of this study was to adapt this technique to equine embryos and to compare the index of DNA replication (S-phase) between equine and caprine embryos. Seventeen equine embryos were recovered at day 6.5 post-ovulation and 20 caprine embryos were recovered at day 7 after the onset of estrus. Equine embryos were incubated during 1h at 39 degrees C in PBS containing 1mM of BrdU. Embryos were then treated in 0.05% trypsin during 15 min ...
Christmann U, Hite RD, Witonsky SG, Elvinger F, Werre SR, Thatcher CD, Tan RH, Buechner-Maxwell VA.Surfactant alterations are described in horses after exercise, anesthesia, and prolonged transport, in horses with recurrent airway obstruction, and in neonatal foals. The effect of horse age or bronchoalveolar lavage fluid (BALF) sample characteristics on surfactant is unknown. Objective: To evaluate surfactant phospholipid composition and function in healthy horses, and to investigate the influence of age and BALF sample characteristics on surfactant. Methods: Seventeen healthy horses 6-25 years of age maintained on pasture year-round. Methods: BALF was collected by standard procedures and w...
Greene CE, Tsang VC, Prestwood AK, Meriwether EA.Optimal reaction conditions for the activated partial thromboplastin time (APTT), 1-stage prothrombin time (PT), and Russell's viper venom time were studied for pooled plasmas of horses, cattle, sheep, goats, swine, dogs, cats, and persons. Changes in CaCl2 and NaCl concentrations had significant effects on the APTT and PT reactions. The APTT was more sensitive than the PT to changes in CaCl2 concentration. The CaCl2 concentration recommended by the manufacturer for the APTT was suboptimal for some of plasmas of domesticated animals in ths study. Infusorial earth (Celite, activator) concentrat...
Schmitz A, Thormann W, Moessner L, Theurillat R, Helmja K, Mevissen M.Ketamine, an injectable anesthetic and analgesic consisting of a racemic mixture of S-and R-ketamine, is routinely used in veterinary and human medicine. Nevertheless, metabolism and pharmacokinetics of ketamine have not been characterized sufficiently in most animal species. An enantioselective CE assay for ketamine and its metabolites in microsomal preparations is described. Racemic ketamine was incubated with pooled microsomes from humans, horses and dogs over a 3 h time interval with frequent sample collection. CE data revealed that ketamine is metabolized enantioselectively to norketamine...
Schellenbacher C, Shafti-Keramat S, Huber B, Fink D, Brandt S, Kirnbauer R.The consistent and specific presence of Equus caballus papillomavirus type 2 (EcPV2) DNA and mRNA in equine genital squamous cell carcinoma (gSCC) is suggestive of an etiological role in tumor development. To further validate this concept, EcPV2-neutralizing serum antibody titers were determined by an EcPV2 pseudovirion (PsV) neutralization assay. Furthermore, an EcPV2 L1 virus-like particle (VLP)-based vaccine was generated and its prophylactic efficacy evaluated in vivo. All 6/6 gSCC-affected, but only 3/20 tumor-free age-matched animals revealed EcPV2-neutralizing serum antibody titers by P...
Yun J, Oyungerel B, Kong HS.This study aimed to identify the genetic diversity and population structure of Mongolian horse populations according to the province of residence (Khentii, KTP; Uvs, USP; Omnogovi and Dundgovi, GOP; Khovsgol, KGP) using 14 microsatellite (MS) markers. Methods: A total of 269 whole blood samples were obtained from the four populations (KTP, USP, GOP, KGP) geographically distinct provinces. Multiplex polymerase chain reaction (PCR) was conducted using 14 MS markers (AHT4, ASB2, ASB17, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, and VHL20), as recommended by the International So...
Manao G, Camici G, Stefani M, Berti A, Cappugi G, Liguri G, Nassi P, Ramponi G.Acylphosphatase was purified from horse muscle by a new procedure involving an affinity chromatography step and subsequent ion-exchange chromatography. This procedure was considerably milder than the preceding one, gave an overall yield of about 60% of activity and permitted isolation of three molecular forms with acylphosphatase activity. All these enzymatic forms are tightly bound to Sepharose 4B-linked anti-horse muscle acylphosphatase antibodies. Two of these forms (Ho1 and Ho3) are present in larger amounts: Ho1 corresponds to the enzyme purified according to the older procedure; this enz...
Bertone AL, Davis DM, Cox HU, Kamerling SS, Roberts ED, Caprile KA, Gossett KA.To evaluate the clinical, laboratory, and histologic effects of 2 methods of treatment for infectious arthritis in horses, Staphylococcus aureus (3.4 to 3.9 x 10(3) colony-forming units) was inoculated into the tarsocrural joints of 8 horses on day 0. Each horse was treated with phenylbutazone (2 g, PO, q 24 h) and gentamicin sulfate (2.2 mg/kg of body weight, IV, q 8 h) for 14 days. On day 2, general anesthesia was induced, and each horse had 1 tarsocrural joint treated by arthrotomy, with removal of accessible fibrin and lavage with 3 L of sterile balanced electrolyte solution. An indwelling...
McD￮ LA, Anderson GI, Wright GM, Ryan DA.Bone cell cultures were evaluated to determine if osteogenic cell populations at different skeletal sites in the horse are heterogeneous. Osteogenic cells were isolated from cortical and cancellous bone in vitro by an explant culture method. Subcultured cells were induced to differentiate into bone-forming osteoblasts. The osteoblast phenotype was confirmed by immunohistochemical testing for osteocalcin and substantiated by positive staining of cells for alkaline phosphatase and the matrix materials collagen and glycosaminoglycans. Bone nodules were stained by the von Kossa method and counted....
Meyers SA, Overstreet JW, Liu IK, Drobnis EZ.Mammalian sperm that have completed capacitation are capable of undergoing the acrosome reaction in response to a number of biological and chemical stimuli. In the present report, we have investigated the ability of progesterone to stimulate acrosome reactions of stallion sperm capacitated in vitro. Motile sperm were selected by a two-layer Percoll gradient centrifugation and were incubated in TALP medium modified by the 1:1 (v/v) addition of TEST-yolk medium for 5 hours at 39 degrees C, under 5% CO2 in humidified air. Sperm incubated in vitro in TALP-TEST medium had a higher percentage of acr...
Golke A, Cymerys J, Słońska A, Dzieciatkowski T, Chmielewska A, Tucholska A, Bańbura MW.Real-time cell electronic sensing (RT-CES) based on impedance measurements is an emerging technology for analyzing the status of cells in vitro. It allows label-free, real time monitoring of the biological status of cells. The present study was designed to assess dynamic data on the cell processes during equine herpesvirus type 1 (EHV-1) infection of ED (equine dermal) cells and primary murine neuronal cell culture. We have demonstrated that the xCELLigence system with dynamic monitoring can be used as a rapid diagnostic tool both to analyze cellular behavior and to investigate the effect of v...
Gautier C, Scarlet D, Ertl R, Walter I, Wulf M, Nagel C, Aurich J, Aurich C.The aim of the present study was to characterise key enzymes involved in polyunsaturated fatty acid (PUFA) synthesis in the testis and epididymis collected from 2-year-old healthy warmblood stallions (n=10). The mRNA expression of fatty acid synthase, the Δ9-, Δ6-, Δ5- and Δ4-desaturases and elongases 6, 5 and 2 (encoded by the fatty acid synthase (FASN), the stearoyl-CoA desaturase (SCD), the fatty acid desaturase 2 (FADS2), the fatty acid desaturase 1 (FADS1), the delta 4-desaturase, sphingolipid 1 (DEGS1), ELOVL fatty acid elongase 6(ELOVL6), ELOVL fatty acid elongase 5 (ELOVL5), ELOVL ...
Ortgies F, Klewitz J, Görgens A, Martinsson G, Sieme H.Reasons for low in vitro fertilisation rates in the horse include the difficulties in inducing capacitation and/or hyperactivation of stallion spermatozoa. The aim of this study was to analyse the effect of noncapacitating and capacitating modified Whitten's (MW) and modified Tyrode's medium (MT) and treatment with procaine (5 mmol), pentoxifylline (3.5 mmol) and trolox (120 mmol) on motility (CASA), capacitation, acrosomal status, viability and mitochondrial membrane potential of stallion spermatozoa (n = 4). While there was no influence of MW and MT on sperm motility, a significant increase ...
Nagaoka K, Nambo Y, Nagamine N, Nagata SI, Tanaka Y, Shinbo H, Tsunoda N, Taniyama H, Watanabe G, Groome NP, Taya K.The relationship between a selective increase in circulating immunoreactive (ir)-inhibin and the time of ovulation was investigated in mares. Concentrations of plasma ir-inhibin were measured every 4 h during the periovulatory period. Inhibin pro-alphaC, a precursor protein of the inhibin alpha-subunit, was also measured. The changes in ir-inhibin and inhibin pro-alphaC in circulation were parallel. Concentrations of both ir-inhibin and inhibin pro-alphaC in the plasma increased at the same time when ovulatory follicles ruptured, and the peak levels of circulating ir-inhibin and inhibin pro-al...
Goudet G, Belin F, Bézard J, Gérard N.The intrafollicular content of LH receptor, alpha-inhibin, and aromatase are known good indicators of follicular status. We investigated the amounts of these proteins in granulosa and cumulus cells in relation to oocyte competence for in vitro maturation, follicular growth, and estrous cycle stage in the mare. Follicular punctures were performed 34 h after an injection of crude equine gonadotropins, either during the follicular phase, at the end of the follicular phase, or during the luteal phase. The cumulus-oocyte complex, granulosa cells, and follicular fluid of follicles larger than 5 mm w...
Cutler C, Viljanto M, Hincks P, Habershon-Butcher J, Muir T, Biddle S.LGD-4033 is one of a number of selective androgen receptor modulators (SARMs) that are being developed by the pharmaceutical industry to provide the therapeutic benefits of anabolic androgenic steroids, without the less desirable side effects. Though not available therapeutically, SARMs are available for purchase online as supplement products. The potential for performance enhancing effects associated with these products makes them a significant concern with regards to doping control in sports. The purpose of this study was to investigate the metabolism of LGD-4033 in the horse following oral ...
Orino K, Saji M, Ozaki Y, Ohya T, Yamamoto S, Watanabe K.The effects of horse serum on the immunoassay of horse ferritin were investigated using two sandwich enzyme-linked immunosorbent assay (ELISA) systems. In System A, affinity-purified antibody to horse spleen ferritin and its conjugate with alkaline phosphatase were used as the first and second antibodies, respectively. In System B, whole antiserum and its conjugate with the enzyme were used. The recoveries of horse spleen ferritin added to horse sera were very low in either system (50-71% in System A; 42-79% in System B). However, heat treatment of the sera at 75 degrees C for 15 min improved ...
Colas C, Garcia P, Popot MA, Bonnaire Y, Bouchonnet S.A method has been developed for the analysis and characterization in equine urine and plasma of iridoid glycosides: harpagide, harpagoside and 8-para-coumaroyl harpagide, which are the main active principles of Harpagophytum, a plant with antiinflammatory properties. The method involves liquid chromatography coupled with positive electrospray ionization mass spectrometry. The addition of sodium or lithium chloride instead of formic acid in the eluting solvent has been studied in order to enhance the signal and to modify the ion's internal energy. Fragmentation pathways and associated patterns ...
McGilvray TA, Knowles EJ, Harris PA, Menzies-Gow NJ.The chemiluminescence (CL) and immunofluorescence (IF) assays yield different results for basal adrenocorticotropin hormone concentrations [ACTH] in pony plasma. It is unclear whether this difference also occurs in basal samples from horses or samples from ponies following thyrotropin-releasing hormone (TRH) stimulation. Objective: To compare the results of [ACTH] analysis by CL and IF methods in basal samples from horses and pony samples following TRH stimulation. Methods: Method comparison. Methods: Plasma [ACTH] was measured concurrently using CL and IF methods in 12 ponies (basal and post-...
Swinburne JE, Hopkins A, Binns MM.The dominant grey coat colour gene of horses has been mapped using a whole genome scanning approach. Samples from a large half-sibling pedigree of Thoroughbred horses were utilized in order to map the grey coat colour locus, G. Multiplex groups of microsatellite markers were developed and used to efficiently screen the horse genome at a resolution of approximately 22 cM, based on an estimated map length for the horse genome of 2720 cM. The grey gene was assigned to chromosome 25 (ECA25), one of the smaller acrocentric horse chromosomes. Based on the current state of knowledge of conserved synt...
Carnevale EM, Metcalf ES.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...
Boermans HJ, Johnstone I, Black WD, Murphy M.Six horses gavaged with a commercial brodifacoum (BDF)-containing bait (Talone) at a dosage of 0.125 mg of BDF/kg of body weight showed weight loss, severe hypocoagulability and hemogram alterations. Four of the horses became depressed and anorectic; one required vitamin K1 therapy. Increases in clotting times were observed at 24 h in the partial thromboplastin time (PTT) followed by the thrombotest (TBT) and one-stage prothrombin time (PT) at 48 h. Elevated mean PTT, PT and TBT were observed from days 4 to 8 (p less than 0.05) with levels returning to pretreatment levels by day 12. Maximum pr...
Collins MD, Lundström T, Welinder-Olsson C, Hansson I, Wattle O, Hudson RA, Falsen E.Phenotypic and phylogenetic studies were performed on four unidentified Gram-positive staining, catalase-negative, alpha-hemolytic Streptococcus-like organisms recovered from the teeth of horses. SDS PAGE analysis of whole-cell proteins and comparative 16S rRNA gene sequencing demonstrated the four strains were highly related to each other but that they did not correspond to any recognised species of the genus Streptococcus. Phylogenetic analysis based on 16S rRNA gene sequences showed the unidentified organisms form a hitherto unknown sub-line within the Streptococcus genus, displaying a clos...
Breuhaus BA, Refsal KR, Beyerlein SL.The purpose of the study reported here was to validate measurement of free thyroxine (fT4) concentration in equine serum by equilibrium dialysis (fT4D), and to compare values with fT4 concentration measured directly and with total T4 (TT4) concentration. The fT4D, fT4, and TT4 concentrations were measured over a range of values in euthyroid horses and horses made hypothyroid by administration of propylthiouracil (PTU). Concentrations of fT4D (<1.8-83 pmol/L) were consistently higher than those of fT4 (<1-40 pmol/L). There was a significant (P < .001) regression of fT4D on fT4 in 503 s...
Hendricks KE, Hansen PJ.Apoptosis is common during spermatogenesis. Here, it was tested whether apoptosis could be induced in sperm after ejaculation. There were several lines of evidence to indicate that sperm are resistant to induction of apoptosis. First, incubation of bull sperm at temperatures characteristic of normothermia (38.5 degrees C) or heat shock (40 and 41 degrees C) for 4h did not increase the proportion of sperm positive for the TUNEL reaction. There was also no reduction in mitochondrial polarity caused by exposure to 40 or 41 degrees C. Incubation at 38.5 degrees C (least-squares mean+/-SEM=4.0+/-1....
Tewari D, Kim H, Feria W, Russo B, Acland H.West Nile virus (WNV) RNA was quantified in WNV infected crows and horses with the help of a real-time reverse transcriptase-PCR assay. A 5' nuclease assay, based on NS5 gene detection with a fluorescent probe was used for quantifying WNV RNA using formalin fixed paraffin embedded tissue specimens. Quantitative detection of WNV RNA showed the presence of a higher amount of the viral RNA in crow tissues compared to equine tissues and these results correlated well with the detection of WNV antigen by immunostaining. In crows, the highest amount of virus was seen in the intestine and in horses in...
Maciel JF, Gressler LT, da Silveira BP, Dotto E, Balzan C, Matter LB, Siqueira FM, de Vargas APC.Data about phylogenetic classification of Escherichia coli colonizing calves, lambs and foals are routinely neglected and restricted to outdated methodologies, even in the context of antimicrobial susceptibility (AS) testing. Thus, the aim of this study was to determine the phylogenetic diversity and the AS profile of E. coli colony-forming units (CFUs) from faecal samples of healthy animals. Five CFUs of E. coli were randomly selected from each faecal culture of calves (n = 13), foals (n = 13) and lambs (n = 13), totalizing 195 CFUs phylo-typed by quadruplex PCR. The AS profile of five ...
Gonzalez-Castro RA, Trentin JM, Carnevale EM, Graham JK.We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose-EDTA-egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk-egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially t...
Welsch U, Schumacher U, Buchheim W, Schinko I, Jenness P, Patton S.A specific secretory product of the lactating mammary gland are triglyceride fat globules which are enveloped by a very complex membrane, the milk fat globule membrane (MFGM). In different mammalian species (man, rhesus monkey, horse, goat, sheep, cow, grey seal, camel, alpaka) the glycoproteins of this membrane have been analyzed by gel electrophoresis, Western blotting and lectin histochemistry. A remarkable intra- and interspecific variability of these glycoproteins has been detected pointing to so far unknown physiological adaptions, which may play a role in the intestine of the new born. ...
