Cell viability refers to the ability of cells to survive and function within their physiological environment. In horses, assessing cell viability is an important aspect of veterinary research, particularly in understanding the effects of various treatments, diseases, and environmental factors on equine cellular health. Techniques such as flow cytometry, trypan blue exclusion, and MTT assays are commonly used to evaluate cell viability in equine studies. These methods help determine the proportion of living cells in a sample, providing insights into cellular responses to different stimuli or conditions. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, applications, and implications of cell viability assessments in equine research.
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 ...
Kirk ES, Squires EL, Graham JK.Assessing the fertilizing potential of a semen sample is important for effective stallion management and for rapid progress in evaluating new cryopreservation technologies. Unfortunately, sperm motility does not estimate fertility well. These experiments established assays to measure cell viability, acrosomal integrity and mitochondrial function for cryopreserved stallion spermatozoa, using flow cytometry, and determined the variability associated with these assays. Correlations between results for these laboratory assays and stallion fertility were also determined. The inter-assay variability...
Aziz DM, Ahlswede L, Enbergs H.The assay of MTT reduction depends on the ability of metabolically active cells to reduce the tetrazolium salt (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) to formazan. This study was conducted to examine and validate of a simple and less costly MTT test in determining equine sperm viability and compare the efficiency of this test with a flow cytometer. Fresh ejaculates from 11 stallions (warm blood) were included in this study. Semen was diluted to 100 million cells/ml in a Hepes 0.1% BSA. The rates of MTT reduction were measured in microtiter plates after incubation for 1 an...
Suh TK, Schenk JL, Seidel GE.Sexing sperm by high-speed flow cytometry subjects them to high pressure. The routine operating pressure of the MoFlo SX flow cytometer for sperm sorting for commercial production has been 50 pounds/square inch (psi), with a standard 70 microm standard nozzle tip. It was hypothesized that lowering the sorting pressure could reduce sperm damage. Therefore, a series of experiments using semen from six bulls, sorted with three MoFlo SX sorters, was conducted to determine optimal pressure. An additional experiment was done with stallion spermatozoa. In Experiment 1, sorting at 30 psi compared to 5...
Eldridge-Panuska WD, di Brienza VC, Seidel GE, Squires EL, Carnevale EM.Experiments were conducted to determine viability of equine embryos in vivo after vitrification. In a preliminary study (Experiment 1), embryos were exposed in three steps to vitrification solutions containing increasing concentrations of ethylene glycol and glycerol (EG/G); the final vitrification solution was 3.4 M glycerol + 4.6 M ethylene glycol in a base medium of phosphate-buffered saline. Embryos were warmed in a two-step dilution and transferred into uteri of recipients. No pregnancies were observed after transfer of blastocysts >300 microm (n = 3). Transfer of morulae or blastocyst...
Jenner F, Edwards RB, Voss JR, Southwood L, Markel MD, Richardson DW.To evaluate the use of hydrothermal ablation of articular cartilage for arthrodesis in horses through investigation of the effects of joint lavage with physiologic saline (0.9% NaCI) solution (80 degrees C) for various treatment times on chondrocyte viability in the articular cartilage of the metacarpophalangeal and metatarsophalangeal joints of cadaveric horse limbs. Sample Population-7 pairs of metacarpophalangeal and 8 pairs of metatarsophalangeal joints from 8 Thoroughbreds. Methods: The horses were euthanatized for reasons unrelated to musculoskeletal disease. On a random basis, 1 joint o...
Benton HP, Esquivel AV, Rice AD, Giri SN.Pirfenidone is under investigation as an anti-inflammatory and anti-fibrotic agent in several organs including lung. Since important features of arthritic conditions include inflammation and long-term damage to articular cartilage, we have investigated whether PD can suppress chondrocyte responses to bacterial lipopolysaccharide (LPS) and interleukin 1 (IL-1); modulators that induce a cascade of inflammatory responses that lead to articular joint tissue damage. PD (0 - 5microM) showed no effect on cell number or viability when incubated with high density primary equine chondrocyte cultures for...
Albrizio M, Guaricci AC, Maritato F, Sciorsci RL, Mari G, Calamita G, Lacalandra GM, Aiudi GG, Minoia R, Dell'Aquila ME, Minoia P.The development of fertilizing ability in sperm cells is associated with changes in the plasma membrane. However, to date the exact nature of sequentially activated primary receptors and channels and the signal transduction pathways derived from these remains elusive. We analyzed the expression and localization of the mu-opioid receptor in equine spermatozoa. A transcript corresponding to the third extracellular loop that selectively binds mu agonists was amplified, sequenced and compared with the known sequences in humans, rats and cattle. The amplification product showed a high degree of nuc...
Malisauskas M, Ostman J, Darinskas A, Zamotin V, Liutkevicius E, Lundgren E, Morozova-Roche LA.In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57 degrees C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to oc...
Pomar FJ, Teerds KJ, Kidson A, Colenbrander B, Tharasanit T, Aguilar B, Roelen BA.The occurrence of pregnancies and births after embryo transfer (ET) of in vivo produced embryos is generally more successful compared to that of embryos produced in vitro. This difference in ET success has been observed when embryos of morphological equal (high) quality were used. The incidence of apoptosis has been suggested as an additional criterion to morphological embryo evaluation in order to assess embryo quality and effectively predict embryo viability. In this study, equine, porcine, ovine, caprine and bovine in vivo and in vitro produced morphologically selected high quality (grade-I...
Mudge MC, Macdonald MH, Owens SD, Tablin F.To compare viability of equine whole blood stored by 4 different methods, and to establish optimal storage protocols for an equine autologous blood donation program. Methods: In vitro study of stored equine whole blood. Animals- Six healthy adult horses. Methods: Blood from each horse was collected into 4 different containers: glass bottles containing acid-citrate-dextrose solution (ACD), plastic bags containing ACD, citrate-phosphate-dextrose (CPD), and CPD with supplemental adenine (CPDA-1). Blood was stored for 5 weeks and sampled at 2-day intervals. Standard hematologic and biochemical var...
Elsheikha HM, Mansfield LS.Sarcocystis neurona has become recognized as the major causative agent of equine protozoal myeloencephalitis (EPM) in the Americas. At least 3 pathogenic species of Sarcocystis, including S. neurona, can be isolated from opossums. Methods are needed to ascertain whether these isolates are viable and capable of causing infections. In this study, the nuclear stain propidium iodide (PI) was used to differentiate between live (viable) and heat-killed (nonviable) S. neurona sporocysts. PI was excluded by live sporocysts but penetrated compromised sporocyst membrane and stained sporozoite nuclei of ...
Gigante A, Bevilacqua C, Cappella M, Manzotti S, Greco F.Articular cartilage defects do not heal. Biodegradable scaffolds have been studied for cartilage engineering in order to implant autologous chondrocytes and help cartilage repair. We tested some new collagen matrices differing in collagen type, origin, structure and methods of extraction and purification, and compared the behavior of human chondrocytes cultured on them. Human chondrocytes were grown for three weeks on four different equine type I collagen matrices, one type I, III porcine collagen matrix and one porcine type II collagen matrix. After 21 days, samples were subjected to histoche...
Schneider N, Lejeune JP, Deby C, Deby-Dupont GP, Serteyn D.Ischaemia and reperfusion are suspected to alter chondrocyte metabolism. Here, we studied the effects of three oxygen (O2) tensions on the viability of equine articular chondrocytes isolated from the cartilage of the distal interphalangeal joint of horses. Chondrocytes were cultured in alginate beads under 1%, 5% or 21% gas phase O2 concentration for 14 days, cellular growth kinetics were measured (n=6), and the cells were observed by light microscopy after staining for necrotic and apoptotic cell detection. For information about the metabolic status, the intracellular adenosine triphosphate (...
Wessel MT, Ball BA.Osmotic stress is an important component of the damage to spermatozoa during cryopreservation. Osmotic injury, due to hyperosmolar freezing extenders, changes in relative solute concentration in the extra cellular medium during freezing and differences in the relative permeabilities of penetrating cryoprotectants, such as glycerol, and water occur when cryopreserved spermatozoa are diluted into isosmotic media or when spermatozoa are placed in the female reproductive tract. The purpose of the study reported here was to evaluate the effect of step-wise dilution for the removal of the permeating...
Bass LD, Denniston DJ, Maclellan LJ, McCue PM, Seidel GE, Squires EL.Equine embryos (n=43) were recovered nonsurgically 7-8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol (n=21). Embryos (300-1000 microm) were measured at five intervals after exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75 and 10 min) to determine changes (%) in diameter over time (+/-S.D.). Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22 degrees C) to -6 degrees C. Straws were then seeded, h...
Pedersen HG, Watson ED, Telfer EE.The effects of ovary holding time and temperature on granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology were investigated through a series of experiments. Three experiments were performed to determine the effect of ovary holding time and temperature on granulosa cell apoptosis. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 2h, (2) at 30 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6 or 6-10h, and (3) granulosa cells were held for 0, 1, 2, 3, 5, 12 or 24h in M199 with Hank's salts at room temperature (suboptimal incubation). Granulosa cell DNA was analysed by ...
Zerbe H, Engelke F, Klug E, Schoon HA, Leibold W.An endometritis model was used to investigate the influence of degenerative endometrial changes (endometrosis) on functional parameters of uterine neutrophils in the horse. Six hours after intrauterine application of recombinant human interleukin-8 (rhIL-8), the uteri of 15 mares were flushed with phosphate-buffered saline. Quantitative and qualitative flow cytometric assays were then made to determine the absolute numbers, viability, phenotype, generation of reactive oxygen species (ROS), and phagocytic activity of immigrated polymorphonuclear neutrophilic granulocytes (PMN). Recombinant hIL-...
Shoemaker RW, Read EK, Duke T, Wilson DG.The feasibility of leaving the ovaries within the peritoneal cavity after laparoscopic coagulation and transection of the ovarian pedicle was assessed in the juvenile horse. Elective ovariectomy was performed on 10 quarter horses, aged 4 to 5 mo, with the fillies in a Trendelenburg position. The mesovarium was isolated, and multiple coagulation and transection cycles were performed until all ovarian attachments had been severed. The ovaries were dropped within the abdomen, and hemostasis of the transected mesovarium was evaluated before closure. The mean surgical time was 33 min (range, 23 to ...
Moussa M, Tremoleda JL, Duchamp G, Bruyas JF, Colenbrander B, Bevers MM, Daels PF.The aim of this study was to evaluate the viability (percentage of dead cells) and the incidence of DNA fragmentation of horse embryos after storage in three different media at 5 degrees C for 6 and 24 h. Forty embryos were stored in Emcare Holding Solution for 6 and 24 h, in Hams'F10 or Vigro Holding Plus for 24 h at 5 degrees C (n = 9-10 per group) and 10 embryos were evaluated immediately after collection. First, embryos were stained, immediately after collection or following storage, to detect dead cells (DAPI) and, subsequently, DAPI-stained embryos were fixed and stained to detect DNA fr...
Bakala A, Karlik W, Wiechetek M.In this study a detailed description of the equine hepatocyte isolation procedure is presented. Livers were obtained from horses slaughtered at the local slaughterhouse. For blood removal and liver preservation the following steps are suggested: perfusion with the oxygenated HBSS (0-2 degrees C, with continuous flow of 500-800 ml/min for 3-6 min), protection from ischemia injury by flushing with ice-cold University of Wisconsin Solution (UW, flow rate of 500-800 ml/min), and finally immersion of the liver lobe in UW solution (2 degrees C) during its transport to the laboratory. For equine isol...
McDï¿® LA, Anderson GI.To compare the osteogenic potential of cancellous bone of conventional graft sites with that of one nonconventional site (fourth coccygeal vertebra) and to investigate the tibial periosteum as a donor site with respect to osteogenic potential. Methods: In vitro osteogenic cell culture system. Methods: Eight adult horses. Methods: Cancellous bone or tibial periosteum was aseptically collected and cut into bone chips or periosteal strips of 1 to 2 mm(3) for primary explant cultures. After 2 weeks, primary tissue cultures that yielded a population of osteogenic cells were counted and subcultured ...
Youn HJ, Lakritz J, Kim DY, Rottinghaus GE, Marsh AE.The purpose of this study was to determine whether alcohol extracts of herbs (Sophora flavescens Aiton, Sinomenium acutum (Thunb.) Rehder and E.H. Wilson, Pulsatilla koreana (Yabe ex Nakai) Nakai ex T. Mori, Ulmus macrocarpa Hance and Torilis japonica (Houtt.) DC.) from South Korea, possess in vitro anti-protozoal activity against cultures of Toxoplasma gondii and Neospora caninum. These herbs have been used as human anti-parasitics in Asian countries for many years. Alcohol extracts of these herbs were serially diluted to final concentrations ranging from 625 to 19.5 ng/ml in media and added ...
Baumber J, Sabeur K, Vo A, Ball BA.The objective of this study was to examine the influence of reactive oxygen species (ROS) on equine sperm capacitation. Motile equine spermatozoa were separated on a discontinuous Percoll gradient, resuspended at 10 x 10(6)ml in Tyrode's medium supplemented with BSA (0.5%) and polyvinyl alcohol (0.5%) and incubated at 39 degrees C for 2h with or without the xanthine (X; 0.1mM)-xanthine oxidase (XO; 0.01 U/ml) system or NADPH (0.25 mM). The importance of hydrogen peroxide or superoxide for capacitation was determined by the addition of catalase (CAT; 150 U/ml) or superoxide dismutase (SOD; 150 ...
Zerbe H, Castilho LF, Engelke F, Mattos RC, Schuberth HJ, Klug E, Leibold W.Sufficient numbers of functionally competent polymorphonuclear neutrophil granulocytes (PMN) seem to be of major importance during the course of equine endometritis. In this study, we wanted to establish a method for cryopreservation of functionally competent neutrophils for an intended local endometritis therapy in mares. The separation of leucocytes by hypotonic lysis of whole blood from clinically healthy mares was superior to the separation by dextrose sedimentation. After suspension of the cells in the cryoprotective solution [equine plasma with 5% (v/v) dimethyl sulphoxide (DMSO)], the l...
Witonsky S, Gogal RM, Buechner-Maxwell V, Ahmed SA.To determine whether immune function can be accurately assessed in blood samples obtained from horses and refrigerated overnight and whether a nonradioactive lymphocyte proliferation assay can be used to evaluate samples obtained from horses. Methods: 224 blood samples from 28 clinically normal adult horses. Methods: Heparinized blood samples were collected. Each sample was divided into 2 equal aliquots. One aliquot was refrigerated overnight to simulate overnight shipping of blood samples, and the other aliquot was evaluated on the day of blood collection. Lymphocytes were isolated and enumer...
Galli C, Lagutina I, Crotti G, Colleoni S, Turini P, Ponderato N, Duchi R, Lazzari G.Several animal species, including sheep, mice, cattle, goats, rabbits, cats, pigs and, more recently, mules have been reproduced by somatic cell cloning, with the offspring being a genetic copy of the animal donor of the nuclear material used for transfer into an enucleated oocyte. Here we use this technology to clone an adult horse and show that it is possible to establish a viable, full-term pregnancy in which the surrogate mother is also the nuclear donor. The cloned offspring is therefore genetically identical to the mare who carried it, challenging the idea that maternal immunological rec...
Petrov R, MacDonald MH, Tesch AM, Van Hoogmoed LM.To determine rate and degree of cooling for the superficial digital flexor tendon (SDFT) during a standard cryotherapy application in horses and evaluate in vitro effects of cooling on survival of tendon cells. Methods: 6 limbs of 5 adult horses and cultured cells obtained from SDFT of 3 adult horses during necropsy. Methods: In vivo data were acquired by use of a thermocouple temperature probe inserted into the SDFT of a forelimb of each standing sedated horse. After baseline temperatures were recorded, a commercial compression splint with circulating coolant was placed on each selected limb,...
Sales F, Ferreira-Silva J, Vieira J, Basto S, Filho P, Rocha J, Moura MT, Oliveira M.Post-diluents could potentially increase semen cryotolerance, but remain poorly explored in horses. The aim was to evaluate the efficiency of post-diluents on frozen-thawed semen viability of two stallions (S1-S2). The cryopreserved semen was thawed at 50°C for 40 seconds. Semen motility and acrosin activity (AA) were determined during the thermo-resistance test (TRT). Progressive motility of S2 semen decreased after 60 and 90 minutes of TRT (TRT60 and TRT90) on the control compared to both post-diluents. The total motility of both S1 and S2 decreased on TRT60 and TRT90 semen control versus b...
Hudson NP, Mayhew IG, Pearson GT.Intracellular microelectrode recordings were made from smooth muscle cells in cross-sectional preparations of equine ileum, superfused in vitro. Membrane potential oscillations and spike potentials were recorded in all preparations, but recordings were made more readily from cells in the longitudinal muscle layer than from cells in the circular layer. The mean (se) resting membrane potential (RMP) of smooth muscle cells in the longitudinal muscle layer was -51.9 (1.2) mV, and the membrane potential oscillations in this layer had a mean amplitude of 4.8 (0.4) mV, a frequency of 9.0 (0.1) cycles...
Zizzadoro C, Belloli C, Badino P, Ormas P.A method for the separation of pure and viable lymphocytes and granulocytes from the same blood sample in horses was reported. By centrifuging equine heparinized blood at 100 xg for 10 min at room temperature (r.t.), the resulting supernatant plasma was an almost pure (97.71 +/- 0.30%; n = 15) suspension of highly viable (98.72 +/- 0.28%) lymphocytes. When sodium citrate was used as an anticoagulant, lymphocyte suspensions collected in the same manner showed lower purity (87.89 +/- 1.59%; n = 9) and higher yields (56.56 +/- 3.89%, n = 9 versus 36.11 +/- 2.23%, n = 15). Where needed, a further ...
Zerbe H, Engelke F, Klug E, Schoon HA, Leibold W.An endometritis model was used to investigate the influence of degenerative endometrial changes (endometrosis) on functional parameters of uterine neutrophils in the horse. Six hours after intrauterine application of recombinant human interleukin-8 (rhIL-8), the uteri of 15 mares were flushed with phosphate-buffered saline. Quantitative and qualitative flow cytometric assays were then made to determine the absolute numbers, viability, phenotype, generation of reactive oxygen species (ROS), and phagocytic activity of immigrated polymorphonuclear neutrophilic granulocytes (PMN). Recombinant hIL-...
Xijier , Mori Y, Fukuoka M, Cairangzhuoma , Inagaki M, Iwamoto S, Yabe T, Kanamaru Y.Native alpha-lactalbumins (α-LA) from equine, bovine, and human milk were not cytotoxic. However, after treatment with trifluoroethanol (TFE), all three α-LAs exhibited cytotoxicity. Toxic potencies were distinctly different among them. Equine α-LA was the most robust, bovine α-LA was moderate, and human α-LA was weak. There were no significant structural changes as between the native and the TFE-treated α-LAs.
Silva DM, Holden SA, Lyons A, Souza JC, Fair S.The aim of the present study was to assess the effect of the addition of docosahexaenoic acid (DHA) on the in vitro quality of cooled and frozen-thawed stallion semen. In Experiment 1, semen from 10 stallions was collected (three ejaculates per stallion). Semen was diluted to 100×106 spermatozoa mL-1 with 0.02mM vitamin E (VE) and 0, 1, 10 or 20ng mL-1 DHA and frozen. Semen was thawed and total motility (TM), rapid progressive motility (PM), acrosome integrity, membrane fluidity and morphology were assessed. In Experiment 2, semen from three stallions was collected (three ejaculates per stall...
Moussa M, Bersinger I, Doligez P, Guignot F, Duchamp G, Vidament M, Mermillod P, Bruyas JF.Vitrification using open pulled straw (OPS) has provided encouraging results with embryos from other species. The aim of this study was to compare the survival of 6.5- and 6.75-day-old equine embryos after OPS vitrification and slow-cooling. Eighteen embryos were frozen using a slow-cooling method. Embryos were placed in modified PBS with increasing glycerol concentration (2.5%, 5%, 7.5% and 10% (v/v) 5 min each). Embryos were loaded into 0.25 ml straws then placed in a programmable freezer and subsequently plunged into liquid nitrogen. After thawing, cryoprotectant was removed by five steps w...
Garland A, Wierenga C, McCrae P, Pearson W.The purpose of this study was to determine anti-inflammatory and/or chondroprotective effects of Equine Omega Complete (EOC) on cartilage explants stimulated with lipopolysaccharide (LPS). Explants were aseptically prepared from the intercarpal joints of 17 market-weight pigs and placed in culture at 37°C for a total of 120 hours. For the final 96 hours, explants were conditioned with a simulated digestion extract of EOC (0, 36 or 180 μL/mL), and for the final 48 hours explants were stimulated with LPS (0 or 15µg/mL). Media was removed and replaced every 24 hours. Samples from the final 48 ...
Pruß D, Oldenhof H, Wolkers WF, Sieme H.The aim of this study was to establish an alginate encapsulation procedure for stallion sperm, and investigate if sperm encapsulation enhances longevity during cold storage and survival after cryopreservation. First, biocompatibility of the compounds needed for encapsulation was tested and factors determining capsule structure were identified. Sperm encapsulation was realized either by depositing droplets (20 µL) of sperm solution supplemented with barium or calcium chloride (10 mM) in alginate solution (0.25%, w/v), or by adding sperm-alginate droplets in solution containing barium or calc...
Gibb Z, Grupen CG, Maxwell WM, Morris LH.The fertility of sex-sorted, cryopreserved stallion sperm must be improved for the sex-sorting technology to be applied commercially. Objective: To optimise the conditions used to liquid store stallion sperm prior to sex-sorting and assess the fertility of sperm following sex-sorting and cryopreservation. Methods: Both in vitro experiment and randomised controlled trial in healthy, client-owned mares. Methods: Stallion ejaculates (n = 9) were diluted in either a skimmed milk (KMT) or BSA (I-BSA) based media to 25 × 106 sperm/ml directly (+SP25) or washed to remove seminal plasma and diluted t...
Zerbe H, Castilho LF, Engelke F, Mattos RC, Schuberth HJ, Klug E, Leibold W.Sufficient numbers of functionally competent polymorphonuclear neutrophil granulocytes (PMN) seem to be of major importance during the course of equine endometritis. In this study, we wanted to establish a method for cryopreservation of functionally competent neutrophils for an intended local endometritis therapy in mares. The separation of leucocytes by hypotonic lysis of whole blood from clinically healthy mares was superior to the separation by dextrose sedimentation. After suspension of the cells in the cryoprotective solution [equine plasma with 5% (v/v) dimethyl sulphoxide (DMSO)], the l...
Umair M, Beitsma M, de Ruijter-Villani M, Deelen C, Herrera C, Stout TAE, Claes A.The cryotolerance of equine blastocysts larger than 300 μm can be improved by aspirating blastocoele fluid prior to vitrification; however, it is not known whether blastocoele aspiration also enables successful slow-freezing. The aim of this study was therefore to determine whether slow-freezing of expanded equine embryos following blastocoele collapse was more or less damaging than vitrification. Grade 1 blastocysts recovered on day 7 or 8 after ovulation were measured (>300-550 μm, n = 14 and > 550 μm, n = 19) and blastocoele fluid was aspirated prior to slow-freezing in 10...
Silva GB, De La Côrte FD, Brass KE, Palma HE, Gallio M, Cantarelli C, Bertolin K, Krause A, Wergutz J, Fontanari Krause LM, Antoniazzi AQ.Chondrocyte health is altered when exposed to local anesthetics, raising concerns as to the long-term effects of local anesthetics intra-articularly for diagnosis and analgesia. To investigate the drug with the lowest toxic potential, the effect of ropivacaine and mepivacaine on chondrocytes was evaluated. Articular cartilage from normal metacarpophalangeal joints of five equine cadaver specimens was used to establish chondrocyte cultures. Following seven days, chondrocytes were exposed to standard culture medium (DMEM), ropivacaine 7.5 mg/ml (ROP7.5), ropivacaine 10 mg/ml (ROP10), mepivacaine...
Connelly C, Norton NA, Hurley DJ, Hart KA, Meichner K, Gogal RM.Peripheral blood is commonly sampled to assess the health status of human and veterinary patients. Venous blood collection is a minimally invasive procedure, and in the horse, the common collection site is the jugular vein. Post blood collection, sample processing for leukocyte enrichment can vary by research laboratory with the potential to yield different effects on the enriched cells and their function. The focus of the present study was to compare a common blood dilution-leukocyte enrichment technique using a Histopaque gradient medium (His) to a modified leukocyte buffy coat syringe-lymph...
Mari G, Bucci D, Love CC, Mislei B, Rizzato G, Giaretta E, Merlo B, Spinaci M.The aim of this study was to compare the effect of presorting centrifugation (cushioned [CC] or single-layer colloid [SLC]), with simple dilution (SD), on the quality of sex-sorted stallion semen before and after sorting and after freezing and thawing. Four ejaculates from each of two fertile stallions were collected 1Â week apart and evaluated for percent total sperm motility (TM), percent viable acrosome-intact sperm (VAI), and DNA quality (percentage of DNA fragmentation index). Freezing caused, independently from CC and SLC treatments, a significant decrease of TM (PÂ <Â 0.05) and VAI (...
Faber E, Tshilwane SI, Van Kleef M, Pretorius A.Expanding on our previous work, this study used transcriptome analysis of RNA sequences to investigate the various factors that contributed to either inducing apoptosis that resulted in cell death or promoting the survival of African horse sickness virus serotype 4 (AHSV4)-infected horse peripheral blood mononuclear cells (PBMC) after 24Â h. Apoptosis is a host defense mechanism that prevents virus replication, accumulation and spread of progeny viruses. AHSV4-infected PBMC were killed via the intrinsic and the perforin/granzyme pathways of apoptosis during the attenuated AHSV4 (attAHSV4) in v...
Redding WR, Booth LC.Equine fibroblasts and Staphylococcus aureus were exposed for 30 minutes to six dilutions of chlorhexidine gluconate, a chlorous acid-chlorine dioxide irrigation solution, a chlorous acid-chlorine dioxide disinfectant, and phosphate buffered saline controls. Cell viability was determined by trypsinizing the cells, staining them with trypan blue, and counting cells that did not take the stain. All fibroblasts were killed when exposed to 1.0% and 0.5% chlorhexidine. The survival rate of fibroblasts increased linearly with decreasing concentrations of chlorhexidine gluconate, with a peak survival...
Richer CL, Romagnano A.Both dynamic G-banding and cell synchronization produced by bromodeoxyuridine (BrdU), were applied to equine chromosomes. BrdU incorporated during the first half of the S-phase is taken up into the R-bands that are early replicating. These bands, which have incorporated BrdU, cannot contract as usual and remain elongated; only the other regions of the chromosome, i.e., the G-bands, contract normally and are sharply defined. BrdU also can be used for cell synchronization. The addition of BrdU in a high concentration, 15 hours before harvest, and its removal 11 hours later, has two effects: init...
Oliveira Spila D, Maranhão RPA, Ocarino NM, de Lima JTB, Melo FG, Boeloni JN, Serakides R.The effects of two concentrations of triiodothyronine (T3; 0.01 and 1,000 nM) on the osteogenic and chondrogenic differentiation abilities of equine adipose-derived mesenchymal stem cells (AD-MSCs) were evaluated. The osteogenic study evaluated the effect of T3 using alkaline phosphatase activity (ALP) assay; cell viability and density; and formation of mineralized nodules at Days 7, 14, and 21 in culture. The chondrogenic study tested the effect of T3 through ALP assay, mitochondrial metabolism, cell density, and periodic acid-Schiff-positive (PAS+) matrix percentage at Days 7 and 14. In bot...
Assumpção TI, Lançoni R, Foschini M, Vieira CS.This study aimed to select high-quality spermatozoa by sperm separation by magnetic activation of the fresh equine semen, compared to density gradient centrifugation and evaluating cell quality after selection. The semen of 10 stallions was collected by the artificial vagina technique. The samples analyzed were: (1) fresh semen; (2) density gradient centrifugation (DGC); (3) separation by magnetic activation (MASS) (nonapoptotic portion NAP); (4) separation by MASS (apoptotic portion-APT). Was analyzed: motility (light microscopy), concentration (Neubauer chamber), semen morphology (humid cham...
Melzer M, Burk J, Guest DJ, Dudhia J.Mesenchymal stromal cells (MSC) isolated form bone marrow and adipose tissue are the most common cells used for cell therapy of orthopedic diseases. MSC derived from different tissues show differences in terms of their proliferation, differentiation potential and viability in prolonged cell culture. This suggests that there may be subtle differences in intracellular signaling pathways that modulate these cellular characteristics. The Rho/ROCK signaling pathway is essential for many cellular functions. Targeting of this pathway by the ROCK inhibitor Y-27632 has been shown to be beneficial for c...
Maia L, Dias MC, de Moraes CN, de Paula Freitas-Dell'Aqua C, da Mota LS, Santiloni V, da Cruz Landim-Alvarenga F.Cryopreservation is a feasible alternative to maintaining several cell lines, particularly for immediate therapeutic use, transportation of samples, and implementation of new in vitro studies. This work parts from the hypothesis that the medium of cryopreservation composed by 90% of conditioned medium (CM) supports cryopreservation of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs), allowing the maintenance of the biological properties for the establishment of cell banks intended for therapeutic use and in vitro studies. Thus, we evaluated the viability, apoptosis...
Owens SD, Burges J, Johns JL, Carrade DD, Galuppo LD, Librach F, Borjesson DL.The therapeutic use of bone marrow-derived mononuclear cells (MNCs) and mesenchymal stem cells for the treatment of soft tissue and orthopedic injuries in equine patients is expanding. After collection, bone marrow must be reduced in volume and depleted of RBCs for immediate therapeutic use or to prepare cells for culture or cryopreservation and storage. The MarrowXpress (MXP) System is an automated, closed, sterile system designed to process human bone marrow samples. Objective: The purpose of this study was to evaluate the capacity of the MXP System to process equine bone marrow to reduce vo...
Squires EL.There has and will continue to be reproductive techniques available that have a positive impact upon the equine breeding industry. This review focuses on semen technologies that have been developed or are in the process of being developed. The use of fluorescent dyes and flow cytometry has provided the researcher and clinician with powerful tools to evaluate several sperm attributes. These procedures have been utilized to evaluate sperm viability, acrosome status, mitochondrial status, DNA integrity and stages of capacitation. Flow cytometry allows several sperm attributes to be evaluated on t...
Leach DH.Cells of the intertubular horn of the stratum medium of the equine hoof wall are joined by three types of junctions. Desmosomes and gap junctions were present in all strata. Septate-like junctions and an intercellular line were seen in the distal stratum spinosum subsequent to extrusion of the contents of membrane-coating granules. At a later stage of keratinization, non-membrane-bound acid phosphatase reaction product appeared to leak into the intercellular space except into areas occupied by septate-like junctions and the intercellular line. It is proposed that the formation of septate-like ...
Guimarães T, Lopes G, Ferreira P, Leal I, Rocha A.Cryopreservation of epididymal spermatozoa is a useful tool to preserve genetic material of valuable stallions after emergency castration or unexpected death. For that, testicles and epididymides are generally sent refrigerated to the laboratory. Collection of epididymal spermatozoa is a simple procedure that reduces the volume of the material to be shipped, and may improve the quality of the chilled epididymal sperm cells. In the present study we compared the characteristics of frozen/thawed epididymal spermatozoa after refrigeration of the epididymis or after direct refrigeration of the exte...
Shibany KA, Tötemeyer S, Pratt SL, Paine SW.Despite reports of the successful isolation of primary equine hepatocytes, there are no published data regarding the successful cryopreservation of these isolated cells. In this study, a detailed description of the procedures for isolation, cryopreservation, and recovery of equine hepatocytes are presented. Furthermore, the intrinsic clearance (Cl) and production of metabolites for three drugs were compared between freshly isolated and recovered cryopreserved hepatocytes. Primary equine hepatocytes were isolated using a two-step collagenase perfusion method, with an average cell yield of 2.47Â...
