In vitro research involving horses refers to the study of equine cells, tissues, or biological molecules outside their normal biological context, typically in controlled laboratory environments. This research approach allows scientists to investigate cellular processes, molecular interactions, and the effects of various treatments without the ethical and logistical complexities of in vivo studies. In vitro studies contribute to understanding equine physiology, pathology, and pharmacology by providing insights into cellular responses to pathogens, drugs, and other stimuli. This page compiles peer-reviewed research studies and scholarly articles that explore various in vitro methodologies and their applications in equine science, including cell culture techniques, molecular assays, and drug efficacy testing.
Vietmeier J, Niedorf F, Bäumer W, Martin C, Deegen E, Ohnesorge B, Kietzmann M.A study was performed to evaluate the use of precision-cut lung slices (PCLS) for studies on the contraction of equine airways. Lungs of 10 horses were taken to prepare PCLS of approximately 250 microm from equine lung tissue using a special microtome. The lung slices were cultured and the enclosed small airways were monitored using a microscope with coupled digital camera, which was used to determine the airway luminal area and diameter from digital images. As indicated by the beating of the ciliated epithelium and reactivity of airways on methacholine challenge, the tissue slices were found ...
Johannessen MK, Skretting G, Ytrehus B, Røed KH.Endochondral bone formation is an important process in development and growth of the skeleton; still many of the mechanisms of growth cartilage remain unknown. The aim of this study was to identify genes specifically expressed in growth cartilage by constructing a subtraction cDNA library of the articular-epiphyseal cartilage complex from neonatal foal. Two hundred and eighty-four differently expressed clones, representing five novel and 37 known genes, were detected by subtraction hybridization. The tissue specificity of these genes was verified by reverse Northern analysis, and tissue distri...
Davis JL, Papich MG, Morton AJ, Gayle J, Blikslager AT, Campbell NB.The purpose of this study was to determine the pharmacokinetics of etodolac following oral and intravenous administration to six horses. Additionally, in vitro cyclooxygenase (COX) selectivity assays were performed using equine whole blood. Using a randomized two-way crossover design, horses were administered etodolac (20 mg/kg) orally or intravenously, with a minimum 3-week washout period. Plasma samples were collected after administration for analysis using high pressure liquid chromatography with ultraviolet detection. Following intravenous administration, etodolac had a mean plasma half-li...
Garré B, van der Meulen K, Nugent J, Neyts J, Croubels S, De Backer P, Nauwynck H.Equine herpesvirus 1 (EHV-1) is an important equine pathogen that causes respiratory disease, abortion, neonatal death and paralysis. Although vaccines are available, they are not fully protective and outbreaks of disease may occur in vaccinated herds. Therefore, there is an urgent need for effective antiviral treatment. For three abortigenic (94P247, 97P70 and 99P96) and three neuropathogenic isolates (97P82, 99P136 and 03P37), the effect of acyclovir, ganciclovir, cidofovir, adefovir, 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP) and foscarnet on plaque number was studied. Addition...
Alhassan A, Govind Y, Tam NT, Thekisoe OM, Yokoyama N, Inoue N, Igarashi I.The sensitivity of LAMP, PCR and in vitro culture methods for the detection of Theileria equi and Babesia caballi was evaluated using tenfold serially diluted culture parasites. On day 1 post-culture, both T. equi and B. caballi parasites could only be observed at 1% parasite dilution from the in vitro culture method, whereas LAMP could detect up to 1 x 10(-3)% of both T. equi and B. caballi parasite dilutions, whilst PCR could detect 1 x 10(-3)% T. equi and 1 x 10(-1)% B. caballi parasite dilutions. On day 7 post-culture, the detection limit for T. equi and B. caballi in the in vitro culture ...
Srivastava K, Singh S, Singh P, Puri SK.RPNI, a combination of three commercially available growth media (RPMI-1640, NCTC-135 and IMDM) has been found to support long term continuous cultivation of 3D7 strain of Plasmodium falciparum in the presence of 10% bovine calf serum. During the present study, the suitability of this medium was evaluated for the development of P. falciparum in the presence of horse, goat and rabbit sera as well as various concentrations of ALBUMAX II. RPNI medium supplemented with 10% bovine calf serum (RPNI-BCS) was used as control. The cultures were maintained in candle jars protocol and parasitaemia was mo...
Portier K, Guichardant M, Debouzy JC, Crouzier D, Geraud I, Kirschvink N, Lekeux P, Fellmann N, Coudert J.Whether direct exposure to different concentrations (0%, 13%, 100%) of oxygen may affect horse erythrocyte membrane fluidity (EMF) and fatty acid (FA) composition was studied during 1 (T60) and 2h (T120) exposure. EMF was investigated at the head group level and hydrophobic core thanks to phosphorus nucleus 31 ((31)P) nuclear magnetic resonance ((31)P NMR) and electronic paramagnetic resonance (EPR) using two spin probes: 5-nitroxydestearic acid and 16-doxylstearic acid. Lipid structure of the membranes was studied by gas liquid chromatography. 4-Hydroxy-2E-nonenal was also analyzed as a marke...
Mills PC.The effects of three vehicles, phosphate-buffered saline (PBS), ethanol (50% in PBS w/w) and propylene glycol (50% in PBS w/w) on in vitro transdermal penetration of testosterone was investigated in the horse. Skin was harvested from the thorax of five Thoroughbred horses after euthanasia and stored at -20 degrees C until required. The skin was then defrosted and placed into Franz-type diffusion cells, which were maintained at approximately 32 degrees C by a water bath. Saturated solutions of testosterone, containing trace amounts of radiolabelled [14C]testosterone, in each vehicle were applie...
Barton MH, Hurley D, Norton N, Heusner G, Costa L, Jones S, Byars D, Watanabe K.Lactoferrin is a colostral glycoprotein with antimicrobial properties. Objective: (1) Serum lactoferrin and immunoglobulin G (IgG) concentrations are correlated and increase in healthy foals after ingestion of colostrum; (2) compared to healthy foals, ill foals will have lower lactoferrin concentrations that correlate with their IgG concentration, neutrophil count, the diagnosis of sepsis, and survival; and (3) plasma concentrations of lactoferrin will be less than serum concentrations. Methods: Healthy foals (n = 16), mature horses (n = 10), and ill foals 1-4 days old (n = 111) that were exam...
Schneeweiss W, Stanek C, Wagner M, Hein I.The potential of five different commercial DNA isolation methods to remove real-time PCR inhibitors from the synovial fluid of horses, cattle and pigs was investigated. All kits with the exception of one included a silica column-based purification of the DNA. With the fifth kit, DNA purification is achieved by removing contaminating macromolecules by a desalting process. We used a recently developed method based on comparison of the real-time PCR signal of an artificial target incorporated into each PCR reaction in the presence of the isolated DNA from the sample, and in control samples contai...
Whalley JM, Ruitenberg KM, Sullivan K, Seshadri L, Hansen K, Birch D, Gilkerson JR, Wellington JE.Equine herpesviruses 1 and 4 (EHV-1 and EHV-4) cause equine respiratory disease worldwide. However, only EHV-1 is a cause of abortion and neurological disease, despite the two viruses having all 76 genes in common. In addition EHV-1 has a broader host range in cell culture than EHV-4, as exemplified by the rabbit kidney (RK) cell line that is permissive for EHV-1, but not for EHV-4. Here we describe that when EHV-4 produced in equine cells was inoculated onto RK cells expressing glycoprotein D of EHV-1 (RKgD1), infection developed as clusters of rounded cells, and this infectivity could be pas...
Ahmed YA, Tatarczuch L, Pagel CN, Davies HM, Mirams M, Mackie EJ.Post-proliferative chondrocytes in growth cartilage are present in two forms, light and dark cells. These cells undergo hypertrophy and die by a mechanism that is morphologically distinct from apoptosis, but has not been characterized. The aims of the current study were to document the ultrastructural appearance of dying hypertrophic chondrocytes, and to establish a culture system in which the mechanism of their death can be examined. Methods: Growth cartilage from fetal and growing postnatal horses was examined by electron microscopy. Chondrocytes were isolated from epiphyseal cartilage from ...
Watts AE, Fubini SL, Vernier-Singer M, Golkowski C, Shin S, Todhunter RJ.To determine the effect of nonthermal plasma on Staphylococcus aureus, fibroblasts in monolayer culture, and clean and contaminated skin explants. Methods: Normal skin from euthanized horses. Methods: S aureus organisms were plated and treated with nonthermal plasma followed by bacterial culture to assess viability. Fibroblasts in monolayer culture and the epidermal and dermal surfaces of clean and S aureus-contaminated skin explants were treated. The effects of distance and duration on the response to treatment were compared. Results: Compared with controls, treatment with nonthermal plasma r...
Lorenzo-Figueras M, Pusterla N, Byrne BA, Samitz EM.To evaluate the effectiveness of a commercial conventional blood culture system (BCS), a commercial resin-containing BCS, and a commercial lysis-centrifugation-based BCS for the recovery of Escherichia coli from equine blood samples inoculated with that organism. Methods: Samples of blood obtained from a clinically normal horse that were inoculated with E coli. Methods: Blood samples were aseptically collected and inoculated with an E coli specimen (50 CFUs/mL) that had been previously isolated from a foal with sepsis. Subsequently, samples were spiked with gentamicin at a concentration of 30 ...
Yates AC, Stewart AA, Byron CR, Pondenis HC, Kaufmann KM, Constable PD.To determine the effects of sodium hyaluronate (HA) in combination with methylprednisolone acetate (MPA) on interleukin-1 (IL-1)-induced inflammation in equine articular cartilage pellets. Methods: Chondrocytes collected from 7 horses euthanatized for problems unrelated to the musculoskeletal system. Methods: Chondrocyte pellets were treated with medium (negative control); medium containing IL-1 (positive control); or medium containing IL-1 with MPA only (0.05 or 0.5 mg/mL), HA only (0.2 or 2 mg/mL), or MPA (0.05 or 0.5 mg/mL) and HA (0.2 or 2 mg/mL) in combination. Proteoglycan (PG) synthesis...
Jones SL, Valenzisi A, Sontakke S, Sprayberry KA, Maggi R, Hegarty B, Breitschwerdt E.Effusive, fibrinous pericarditis is an uncommon disease entity in horses. In 2001, pericarditis occurred in conjunction with an epizootic in central Kentucky that was associated with exposure to eastern tent caterpillars (ETCs). Bacterial isolation from equine pericardial fluid samples was attempted using an insect cell culture growth medium (ICCGM). Using previously cultured, stored frozen samples from four horses with fibrinous pericarditis, inoculation of 10% blood agar plates yielded no growth, whereas simultaneous inoculation of ICCGM resulted in the isolation of Proprionibacterium acnes,...
Nakagawa K, Tokushima A, Fujiwara K, Ikeguchi M.The secondary structure in the molten globule state (an equilibrium analogue of a burst-phase folding intermediate) of equine beta-lactoglobulin was investigated by changes in the circular dichroic spectrum induced by a series of site-directed proline substitutions. The results challenge the structural picture obtained from previous hydrogen/deuterium exchange experiments. A stable non-native alpha-helix was found to exist in the region corresponding to the eighth strand (H strand) in the native structure, where the backbone amide protons are the most strongly protected from exchange. Therefor...
Kútvölgyi G, Stefler J, Kovács A.A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs....
Bruemmer JE.The ability to harvest and preserve epididymal sperm from a stallion after simple elective castration, a catastrophic injury, or severe acute illness and subsequent death has been realized, allowing for the preservation of genetics that would have been lost otherwise.Currently, the care taken to collect the testes and epididymides properly, coupled with proper packaging and shipping, could make the greatest contribution to salvaging viable sperm. As advances in assisted reproductive techniques continue, more offspring may be obtained from stored epididymal sperm from valuable stallions.
Minervini F, Giannoccaro A, Fornelli F, Dell'Aquila ME, Minoia P, Visconti A.The mycotoxin zearalenone (ZEA) and its derivatives, alpha and beta-zearalenol (alpha and beta-ZOL), synthesized by genera Fusarium, often occur as contaminants in cereal grains and animal feeds. The importance of ZEA on reproductive disorders is well known in domestic animals species, particularly in swine and cattle. In the horse, limited data are available to date on the influence of dietary exposure to ZEA on reproductive health and on its in vitro effects on reproductive cells. The aim of this study was to evaluate the effects of ZEA and its derivatives, alpha and beta-ZOL, on granulosa c...
Apprich V, Spergser J, Rosengarten R, Stanek C.The effects of two dermatophytes (Microsporum gypseum and Trichophyton mentagrophytes) and four moulds (Scopulariopsis brevicaulis, Alternaria alternata, Geotrichum candidum and Penicillium spp.) on living keratinocyte cultures were examined in vitro using primary human keratinocytes. Rates of apoptosis of infected cells were determined using a colorimetric TUNEL system which detects the characteristic nuclear DNA fragmentation of apoptotic cells. The cytotoxicity of the individual fungi was tested by quantitatively measuring cytosolic enzyme lactate dehydrogenase, released upon cell lysis, in...
Pain R, Sneddon JC, Cochrane CA.Franz diffusion cells containing uniformly dehydrated equine blood clots to simulate fibrinous eschar were used to measure the rate of debridement of fibrin by novel and traditional wound dressings, under standardised conditions of temperature, pH and humidity. Significant increases in protein breakdown occurred within 24 hours with all the dressings, but not thereafter. In general, dressings hydrated in normal saline were better as debriding agents than dressings hydrated in water. Autolytic debriding agents were 47 per cent more effective than chemical debriding agents; specifically, hydrofi...
Schnabel LV, Mohammed HO, Miller BJ, McDermott WG, Jacobson MS, Santangelo KS, Fortier LA.Platelet rich plasma (PRP) has recently been investigated for use in tissue regeneration studies that seek to utilize the numerous growth factors released from platelet alpha-granules. This study examined gene expression patterns, DNA, and collagen content of equine flexor digitorum superficialis tendon (SDFT) explants cultured in media consisting of PRP and other blood products. Blood and bone marrow aspirate (BMA) were collected from horses and processed to obtain plasma, PRP, and platelet poor plasma (PPP). IGF-I, TGF-beta1, and PDGF-BB were quantified in all blood products using ELISA. Ten...
Hosaka Y, Ozoe S, Kirisawa R, Ueda H, Takehana K, Yamaguchi M.The aim of this study was to clarify whether matrix metalloproteinases (MMP-2 and -9: gelatinases) and pro-inflammatory cytokines [tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta] are induced by heat in tendon tissue in vitro and to test the hypothesis that heat exposure causes tendinocytes to synthesize pro-inflammatory cytokines and that synthesis of these cytokines, in turn, leads to up-regulation of synthesis of gelatinases. Isolated tendinocytes from equine superficial digital flexor tendons were cultured and all experiments were performed on cells passaged 3 or 4 times. In t...
Stanley SM, Wee WK, Lim BH, Foo HC.Direct-injection LC-LC hybrid tandem MS methods have been developed for undertaking broad-based screening for acidic drugs in protein-precipitated plasma and neutral doping agents in equine urine. In both analyses, analytes present in the matrix were trapped using a HLB extraction column before being refocused and separated on a Chromolith RP-18e monolithic analytical column using a controlled differential gradient generated by proportional dilution of the first column's eluent with water. Each method has been optimised by the adoption of a mobile phase and gradient that was tailored to enhanc...
Womble A, Giguère S, Murthy YV, Cox C, Obare E.The objectives of this study were to determine the serum and pulmonary disposition of tilmicosin in foals and to investigate the in vitro activity of the drug against Rhodococcus equi and other common bacterial pathogens of horses. A single dose of a new fatty acid salt formulation of tilmicosin (10 mg/kg of body weight) was administered to seven healthy 5- to 8-week-old foals by the intramuscular route. Concentrations of tilmicosin were measured in serum, lung tissue, pulmonary epithelial lining fluid (PELF), bronchoalveolar lavage (BAL) cells, and blood neutrophils. Mean peak tilmicosin conc...
Milligan M, Kukanich B, Beard W, Waxman S.Lidocaine is administered as an intravenous infusion to horses for a variety of reasons, but no study has assessed plasma lidocaine concentrations during a 12-h infusion to horses. The purpose of this study was to evaluate the plasma concentrations and pharmacokinetics of lidocaine during a 12-h infusion to postoperative horses. A second purpose of the study was to evaluate the in vitro plasma protein binding of lidocaine in equine plasma. Lidocaine hydrochloride was administered as a loading dose, 1.3 mg/kg over 15 min, then by a constant rate IV infusion, 50 microg/kg/min to six postoperativ...
Andrews FM, Buchanan BR, Smith SH, Elliott SB, Saxton AM.To compare the effects of hydrochloric acid (HCl) and various concentrations of volatile fatty acids (VFAs) on tissue bioelectric properties of equine stomach nonglandular (NG) mucosa. Methods: Gastric tissues obtained from 48 adult horses. Methods: NG gastric mucosa was studied by use of Ussing chambers. Short-circuit current (Isc) and potential difference (PD) were measured and electrical resistance (R) and conductance calculated for tissues after addition of HCl and VFAs (5, 10, 20, and 40 mM) in normal Ringer's solution (NRS). Results: Mucosa exposed to HCl in NRS (pH of 1.5 and, to a less...
Lagutina I, Lazzari G, Duchi R, Turini P, Tessaro I, Brunetti D, Colleoni S, Crotti G, Galli C.Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and sign...
Milner PI, Fairfax TP, Browning JA, Wilkins RJ, Gibson JS.To determine the effects of varying O(2) on pH homeostasis, based on the hypothesis that the function of articular chondrocytes is best understood at realistic O(2) tensions. Methods: Cartilage from equine metacarpophalangeal/tarsophalangeal joints was digested with collagenase to isolate chondrocytes, and then loaded with the pH-sensitive fluorophore 2',7'-bis-2-(carboxyethyl)-5(6)-carboxylfluorescein. The radioisotope(22)Na(+) was used to determine the kinetics of Na(+)/H(+) exchange (NHE) and the activity of the Na(+)/K(+) pump, and ATP levels were assessed with luciferin assays. Levels of ...
Yan W, Shin KS, Wang SJ, Xiang H, Divers T, McDonough S, Bowman J, Rowlands A, Akey B, Mohamed H, Chang YF.Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized...
Berreta A, Burbick CR, Alexander T, Kogan C, Kopper JJ.Probiotics are defined as live microorganisms that confer a health benefit to the host when administered in adequate amounts, therefore the presence of viable microorganisms is essential. Previous studies reported inconsistencies when comparing contents of probiotics marketed for use in animals with label claims. However, to the author's knowledge the variability of the same product between different lots has yet to be evaluated. The objective of this observational study was to evaluate the content and inter-lot variability of commercial equine probiotic products using culture dependent and in...
Kisiday JD, Colbath AC, Tangtrongsup S.Ex vivo induction of chondrogenesis is a promising approach to improve upon the use of bone marrow mesenchymal stem cells (MSCs) for cartilage tissue engineering. This study evaluated the potential to induce chondrogenesis with days of culture in chondrogenic medium for MSCs encapsulated in self-assembling peptide hydrogel. To simulate the transition from preconditioning culture to implantation, MSCs were isolated from self-assembling peptide hydrogel into an individual cell suspension. Commitment to chondrogenesis was evaluated by seeding preconditioned MSCs into agarose and culturing in the ...
Bukowiecki CF, Bramlage LR, Gabel AA.Forty-eight limbs of 12 freshly euthanized horses were used to generate data on the strength of the equine suspensory apparatus. The point of failure of the suspensory apparatus of each limb was determined. Immediately before euthanasia, 6 of the 12 horses (thoroughbreds and standardbreds) had been engaged in active training or racing, and six horses in stall and/or pasture activity. In the actively training or racing horses, the point of acute failure of the suspensory apparatus was within the proximal sesamoid bones in 20 (83%) limbs (resulting in 17 apical fractures, 2 basilar fractures, an...
Carnevale EM, Squires EL, McKinnon AO.Forty equine embryos collected 7 d post-ovulation were stored at 5 C for 24 h in one of two culture media (n = 20/group): 1) Ham's F10 + 10% heat-treated fetal calf serum (FCS) buffered by gassing with 5% CO2, 5% O2 and 90% N2 and 2) Ham's F10 + 10% FCS with Hepes buffer (25 mM). Embryos cultured in Ham's F10 + CO2 maintained a better quality score and had a larger average increase in diameter (+34.8 micron) than embryos stored in Hepes buffered Ham's F10 (-10.2 micron). Embryos were transferred surgically into recipient mares that ovulated -3 to +1 d in relation to the donor mare. Twenty embr...
Boscan P, Rezende ML, Grimsrud K, Stanley SD, Mama KR, Steffey EP.To define the pharmacokinetic profile of propofol 5% microemulsion formulation in horses. Methods: First, propofol was administered as bolus injection (2 mg kg(-1)) to six xylazine-sedated horses. Secondly, after sedation and bolus injection, propofol was maintained with continuous infusion for 3 h [8.1 (sd 3.2) mg kg(-1) h(-1)] to the same six horses. Thirdly, in two horses, a commercial propofol was used for comparison. Response to noxious stimulation was used to evaluate analgesia. Venous blood samples were obtained to measure propofol plasma concentration using liquid chromatography-mass s...
Cuervo-Arango J, Claes AN, Stout TA.Asynchronous embryo transfer is an excellent tool to investigate how subtle differences in the uterine environment affect embryo development and survival. Progesterone secreted from the corpus luteum following ovulation is one of the main factors responsible for establishing endometrial receptivity for the pre-implantation embryo via complex alterations in the expression of genes involved in the secretion of the histotroph. The objective of this retrospective study was to determine whether the recipient's Day after ovulation and the number of CL at ET influence the pregnancy rates of IVP horse...
Hoffmann KL, Wood AK, McCarthy PH.Sonographic and anatomic observations were made of the kidneys of 23 Thoroughbreds or Standardbreds. In an in vitro study of 16 horses, precise correlations were established between the gross anatomic features of the kidneys and their sonographic appearance in images obtained in dorsal, sagittal, transverse, and transverse oblique anatomic planes. The renal cortex had a uniformly mottled echogenicity, and the renal medulla was relatively hypoechogenic, compared with the cortex. Acoustic anisotropy was observed in the cortex and medulla of the cranial and caudal extremities of each kidney. The ...
Edmond AJ, Brinsko SP, Love CC, Blanchard TL, Teague SR, Varner DD.Centrifugal fractionation of semen is commonly done to improve quality of human semen in assisted-reproduction laboratories, allowing sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, promoting their retention above defined density media, with structurally normal sperm passing through the media following centrifugation. Three experiments were conducted to evaluate the effects of density-medium type, centrifuge-tube size, sperm number, and density-medium volume (column height) on stallion sperm quality and recovery rate in sperm pelle...
Looze Y, Polastro E, Deconinck M, Leonis J.Spectrophotometric studies of the alkaline isomerization of horse heart and yeast cytochrome c show that the haemoproteins from Saccharomyces cerevisiae differ significantly from the mammalian cytochrome c. Apparent pKa values of 8.41, 8.40 and 8.73 for isol-1-(the methylated and unmethylated forms) and iso-2-cytochrome c respectively, from baker's yeast were determined and compared with the value of 9.40 found for horse heart cytochrome c. The transitions, measured by observing the decrease of the absorbance at 695 nm as the pH increases, have been found to strictly parallel the decrease in a...
Siemieniuch MJ, Kozdrowski R, Szóstek-Mioduchowska AZ, Raś A, Nowak M.Endometritis is one of the most important causes of infertility in mares. Mares may suffer from endometritis outside the breeding season; however, pathological condition is often undiagnosed in anestrus. The aim of this study was to examine whether the secretion profiles of prostaglandin F2α (PGF2α), prostaglandin E2 (PGE2), and a metabolite of prostacyclin I2 (6-keto-PGF1α) differ in endometrial tissue cultures of heavy draft mares in anestrus with endometritis compared to those without endometritis. The endometrial biopsies were collected from 51 heavy draft mares. Inclusion criteria for ...
Nieto JE, Maher O, Stanley SD, Larson R, Snyder JR.To determine the effects of domperidone on in vivo and in vitro measures of gastrointestinal tract motility and contractility in healthy horses. Methods: 18 adult horses and tissue samples from an additional 26 adult horses. Methods: Domperidone or placebo paste was administered to healthy horses in a 2-period crossover study. Gastric emptying was evaluated after oral administration of domperidone paste (1.1 or 5.0 mg/kg) or placebo paste by means of the acetaminophen absorption test in 12 horses. Frequency of defecation, weight of feces produced, fecal moisture, and stomach-to-anus transit ti...
Nakagawa K, Yamada Y, Fujiwara K, Ikeguchi M.Equine beta-lactoglobulin forms a compact intermediate at an acidic pH (A state). It also forms an expanded and helical conformation at low temperatures (C state). The structure of a single disulfide mutant C66A/C160A is similar to the A state in the presence of salts, while it is similar to the C state at low anion concentrations. We have investigated the temperature-dependent change in the secondary structure using circular dichroism and proline scanning mutagenesis. At low anion concentrations, the helical content increased linearly as temperature decreased. In the presence of salts, the A ...
Castaneda C, Radović L, Felkel S, Juras R, Davis BW, Cothran EG, Wallner B, Raudsepp T.Structural rearrangements like copy number variations in the male-specific Y chromosome have been associated with male fertility phenotypes in human and mouse but have been sparsely studied in other mammalian species. Here, we designed digital droplet PCR assays for 7 horse male-specific Y chromosome multicopy genes and SRY and evaluated their absolute copy numbers in 209 normal male horses of 22 breeds, 73 XY horses with disorders of sex development and/or infertility, 5 Przewalski's horses and 2 kulans. This established baseline copy number for these genes in horses. The TSPY gene showed the...
Ahern BJ, Boston RC, Parente EJ.Mechanical evaluation of a novel alternate laryngoplasty system (ALPS). Methods: Experimental. Methods: Cadaveric horse larynges (n = 10). Methods: Arytenoid and cricoid cartilages were embedded for testing. A novel laryngoplasty system consisting of a metallic anchor, 2 stands of #2 suture material, and a metallic button were evaluated. A traditional laryngoplasty using a single strand of #5 Ethibond was evaluated in parallel. Constructs were evaluated in cyclic testing oscillating from 30 to 50 N for 3600 cycles. Constructs subsequently underwent a single cycle to failure. Distraction during...
Roasa LM, Choi YH, Love CC, Romo S, Varner DD, Hinrichs K.In vitro fertilization (IVF) was performed on in vitro-matured equine oocytes in three experiments. Frozen-thawed sperm were prepared using swim-up separation and heparin treatment. In Experiment 1, fertilization was achieved with sperm from only one frozen ejaculate of four obtained from the same stallion. Within this ejaculate, fertilization rates were higher with fresh media, as compared to media held for 6-8 days before use (39.6% versus 7.3%, respectively; P<0.001). The type of bovine serum albumin used affected fertilization rates (4% versus 39.6%; P<0.001). To determine if IVF rates wer...
Song K, Yang GM, Han J, Gil M, Dayem AA, Kim K, Lim KM, Kang GH, Kim S, Jang SB, Vellingiri B, Cho SG.Flavonoids form the largest group of plant phenols and have various biological and pharmacological activities. In this study, we investigated the effect of a flavonoid, 3, 4'-dihydroxyflavone (3, 4'-DHF) on osteogenic differentiation of equine adipose-derived stromal cells (eADSCs). Unassigned: Treatment of 3, 4'-DHF led to increased osteogenic differentiation of eADSCs by increasing phosphorylation of ERK and modulating Reactive Oxygen Species (ROS) generation. Although PD98059, an ERK inhibitor, suppressed osteogenic differentiation, another ERK inhibitor, U0126, apparently increased osteoge...
Ribeiro MG, Pereira TT, de Lima Paz PJ, de Almeida BO, Cerviño CSA, Rodrigues CA, Santos GTS, de Souza Freire LM, Portilho FVR, Filho MFÁ....Central nervous system (CNS) infections comprise life-threatening clinical conditions in domestic species, and are commonly related to severe sequelae, disability, or high fatality rates. A set of bacterial pathogens have been identified in central nervous infections in livestock and companion animals, although the most of descriptions are restricted to case reports and a lack of comprehensive studies involving CNS-related bacterial infections have been focused on a great number of domestic species. In this scenario, we retrospectively investigated selected epidemiological data, clinical findi...
Ferrari M, Weller R, Pfau T, Payne RC, Wilson AM.The purpose of this work was to evaluate the accuracy and precision of a freehand three-dimensional (3-D) ultrasonography system in the determination of lesion volume in tendons. The accuracy and precision of a 3-D ultrasonography system was assessed by performing repeated measurements on a phantom of known volume. Volume measurements of tendon lesions performed with 3-D ultrasonography were compared with measurements based on a series of two-dimensional (2-D) ultrasound (US) scans and to direct measurements from dissections. A novel method for the creation of tendon lesions in vitro was devel...
Mizukami T, Abe Y, Maki K.In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluore...
Mesoangioblasts (MABs) are vessel-associated stem cells that express pericyte markers and are originally isolated from the embryonic dorsal aorta. From postnatal small vessels of skeletal muscle and heart, it is possible to isolate cells with similar characteristics to embryonic MABs. Adult MABs have the capacity to self-renew and to differentiate into cell types of mesodermal lineages upon proper culture conditions. To date, the origin of MABs and the relationship with other muscle stem cells are still debated. Recently, in a phase I-II clinical trial, intra-arterial HLA-matched MABs were pro...
Pickles KJ, Brooks AC, Rickards KJ, Cunningham FM.N-terminal peptides derived from the anti-inflammatory peptide, annexin-1, inhibit neutrophil function but can also induce pro-inflammatory effects. Although equine annexin-1 has been sequenced, its cellular expression and properties have not been reported. This study has examined whether annexin-1 is present in equine leucocytes and how the N-terminal peptide, Ac2-26, affects equine neutrophil superoxide production. Annexin-1 expression in equine neutrophils and mononuclear cells and the ability of Ac2-26 to activate neutrophil p42/44 MAPK were determined by immunoblotting. Equine neutrophil ...
Godovac-Zimmermann J, Conti A, Liberatori J, Braunitzer G.beta-Lactoglobulin isolated from horse colostrum is heterogeneous and contains two components: beta-lactoglobulin I and beta-lactoglobulin II. These two proteins are monomeric and show differences in their electrophoretic mobilities, chain lengths and primary structures. The complete amino-acid sequence of beta-lactoglobulin II was determined by automated Edman degradation of the intact protein and of the peptides derived from these by digestion with trypsin or chymotrypsin and by chemical cleavage with cyanogen bromide. Unlike other beta-lactoglobulins which contain 162 amino acids, horse bet...
Liu Q, Wang XF, Ma J, He XJ, Wang XJ, Zhou JH.Human immunodeficiency virus (HIV)-1 has a unique integration profile in the human genome relative to murine and avian retroviruses. Equine infectious anemia virus (EIAV) is another well-studied lentivirus that can also be used as a promising retro-transfection vector, but its integration into its native host has not been characterized. In this study, we mapped 477 integration sites of the EIAV strain EIAVFDDV13 in fetal equine dermal (FED) cells during in vitro infection. Published integration sites of EIAV and HIV-1 in the human genome were also analyzed as references. Our results demonstrat...
van den Belt AJ, Keg PR, Kik KJ, Barneveld A.An in vitro study was carried out to define the correlation between the dose and distribution of intratendinous fluid injections in the different flexor tendons/ligaments of the palmar/plantar, metacarpal/metatarsal region of the horse. Injection of 0.1-0.2 ml resulted in a local intratendinous fluid depot showing minimal abaxial spreading and more extensive proximodistal expansion. If 0.3-0.5 ml was injected the abaxial spreading remained minimal but the proximodistal expansion increased significantly, the depots in the axial proximal, mid and distal region flowing together. Also minimal peri...
Belacic ZA, Sullivan SN, Rice HC, Durgam SS.To investigate the effects of interleukin-1β (IL-1β) and methylprednisolone acetate (MPA) on equine intrabursal deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) cells in vitro. Methods: Third passage DDFT and NBF cells from 5 healthy donor horses ages 11-17 years euthanized for reasons unrelated to musculoskeletal conditions. Methods: Aggregate cultures were incubated with culture medium alone (control), 10 ng/mL IL-1β, 10 ng/mL IL-1β + 0.05 mg/mL MPA, or 10 ng/mL IL-1β + 0.5 mg/mL MPA for 24 hours. Extracellular matrix (ECM) gene expressions were assessed via rea...
Lima-Neto JF, Fernandes CB, Alvarenga MA, Golim MA, Landim-Alvarenga FC.This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and pro...
Vendrig JC, Coffeng LE, Fink-Gremmels J.Dietary supplementation with oligosaccharides has been proven to be beneficial for health in several mammalian species. Next to prebiotic effects resulting in a modulation of gut micro biota, immunomodulatory effects of oligosaccharides have been documented in vivo. Supplementation with defined oligosaccharide fractions has been shown to attenuate allergic responses and enhance defensive immune responses. Despite the accumulating evidence for immunomodulatory effects, very limited information is available regarding the direct mechanism of action of oligosaccharides. This study aims to elucidat...
Martin LM, Johnson PJ, Amorim JR, Honaker AR, Donaldson RS, DeClue AE.Horses affected with gastrointestinal conditions such as colic or colitis are at substantial risk for translocation of bacterial components such as lipopolysaccharide (LPS, endotoxin) from the gastrointestinal tract into circulation resulting in systemic inflammation and subsequent morbidity and mortality. Therefore, there is a need for effective preventive and treatment strategies aimed at minimizing the host's inflammatory reaction to these pathogen-associated molecular patterns (PAMPs) from gastrointestinal disease. Resveratrol (RES, trans-3,5,4'-trihydroxystilbene) is a phytoalexin commonl...
Shahrabadi MS, Marusyk RG, Crawford TB.Sequential changes induced by an equine adenovirus in cultured fetal equine kidney cells were studied by electron microscopy. The first morphological change was the appearance of type I inclusions. These inclusions developed to type II inclusions which appeared as ring forms. Type III inclusions were formed within the central part of type II inclusions and finally filled up most of the nuclear space. As the infection proceeded, type IV inclusions which appeared as dense dark-staining spheres were formed at the center of the type III inclusions and also inside the cytoplasm. These dark-staining...
