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Topic:Cell Viability
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.
Physiological death of hypertrophic chondrocytes. 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 ...
In vitro analysis of nonthermal plasma as a disinfecting agent. 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...
Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa. 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....
Effect of heat on synthesis of gelatinases and pro-inflammatory cytokines in equine tendinocytes. 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...
Effect of seminal plasma fractions on stallion sperm survival after cooled storage. This study aimed to evaluate stallion sperm survival after 24 h of cooled storage in the presence of seminal plasma (SP) derived from the sperm-rich fractions (SRF) or sperm-poor fractions(SPF) of the ejaculate, without SP, or in the presence of SP from other stallions. Ejaculates were collected from four stallions using an automated phantom, which separated the semen into five cups. Centrifuged and washed spermatozoa from cup 2 (SRF) were mixed with skim milk extender to a concentration of 100 x 10(6) sperm/ml and then 1:1 (v/v) with SP from the stallion's own or another stallions' second (SP...
Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium. Low-level production of the superoxide anion (O2*-) is an important signal transduction event in sperm function including capacitation; however, excessive production of O2*- can be detrimental to sperm function. The objective of this study was to assess dihydroethidium (DHE) as a probe for O2*- in equine spermatozoa. Ejaculated spermatozoa were separated by centrifugation over a Percoll gradient (40:80), and loaded with DHE (2.0 microM) as well as with calcein-acetoxymethylester (CAM, 7.8 nM) to determine cell viability. In Experiment 1, cells were incubated with the xanthine-xanthine oxidase ...
Comparison of mechanical debridement and radiofrequency energy for chondroplasty in an in vivo equine model of partial thickness cartilage injury. The purpose of this study was to develop a long-term model of cartilage injury that could be used to compare the effects of radiofrequency energy (RFE) and mechanical debridement as a treatment. Methods: Partial thickness fibrillation of patellar cartilage was created in 16 mature ponies. Three months after the initial surgery all injured patellae were randomly selected to receive one of the four treatments (n = 8/treatment): (1) control, (2) mechanical debridement with a motorized shaver, (3) TAC-CII RFE probe, and (4) CoVac 50 RFE probe. The ponies were euthanized 22 months after treatment. ...
Cryopreservation of horse semen under laboratory and field conditions using a Stirling Cycle freezer. A Stirling Cycle freezer has been developed as an alternative to conventional liquid nitrogen controlled rate freezers. Horse semen samples were cooled in 0.25 ml straws and 15 ml bags in the Stirling Cycle freezer under laboratory conditions and as a portable device, powered from a car battery. For comparison, straws were frozen in a conventional liquid nitrogen controlled rate freezer. Upon thawing, motility and viability of samples frozen in the Stirling Cycle freezer were not significantly different when compared to samples frozen in the liquid nitrogen freezer. Unlike liquid nitrogen syst...
Evaluation of permissiveness and cytotoxic effects in equine chondrocytes, synovial cells, and stem cells in response to infection with adenovirus 5 vectors for gene delivery. To evaluate host cell permissiveness and cytotoxic effects of recombinant and modified adenoviral vectors in equine chondrocytes, synovial cells, and bone marrow-derived mesenchymal stem cells (BMD-MSCs). Methods: Articular cartilage, synovium, and bone marrow from 15 adult horses. Methods: Equine chondrocytes, synovial cells, and BMD-MSCs and human carcinoma (HeLa) cells were cultured and infected with an E-1-deficient adenovirus vector encoding the beta-galactosidase gene or the green fluorescent protein gene (Ad-GFP) and with a modified E-1-deficient vector with the arg-gly-asp capsid pepti...
Rapid, multiwell colorimetric assay for measuring neutrophil chemoattractant activity in bronchoalveolar lavage fluid of horses with recurrent airway obstruction. The criteria used to diagnose recurrent airway obstruction (RAO) in affected horses include demonstration of reversible lower airway obstruction and greater than 25% neutrophils in bronchoalveolar lavage fluid (BALF). Additional objective laboratory tests are needed to improve diagnostic accuracy and to monitor response to treatment. The goal of this study was to determine if neutrophil chemoattractant activity of BALF could be measured by using a previously described, rapid, multiwell colorimetric assay for chemotaxis. In this assay, neutrophils that have migrated through a membrane filter ar...
Production of horse foals via direct injection of roscovitine-treated donor cells and activation by injection of sperm extract. We evaluated the effects of different donor cell treatments and activation methods on production of blastocysts after equine nuclear transfer. Nuclear transfer was performed by direct injection of donor cells, using a piezo drill, and standard activation was by injection of sperm factor followed by culture with 6-dimethylaminopurine. There was no difference in blastocyst development between embryos produced with roscovitine-treated or confluent donor cells (3.6% for either treatment). Addition of injection of roscovitine or culture with cycloheximide at the time of activation did not affect bl...
Intermediate amyloid oligomers of lysozyme: Is their cytotoxicity a particular case or general rule for amyloid? In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomers of horse milk lysozyme. We have shown that lysozyme forms soluble amyloid oligomers and protofibrils during incubation at pH 2.0 and 4.5 and 57 degrees C. These structures bind the amyloid-specific dyes thioflavin T and Congo Red, and their morphology and size were analyzed by atomic force microscopy. Monomeric lysozyme and its fibrils did not affect the viability of three cell types used in our experiments including primary murine neurons and fibroblasts, as well as neuroblastoma cell line IMR-3...
New staining methods for sperm evaluation estimated by microscopy and flow cytometry. New staining methods and automated instruments are now available to evaluate the sperm cell in vitro. Individual compartments of the sperm cell, such as the nucleus and the plasma and acrosomal membranes, may be investigated, as well as the cell function as shown by mitochondria activity and capacitation. Various probes are used and they can be analyzed by direct light or fluorescent microscopy or by flow cytometry. The automated instruments allow objective and accurate analysis and quantification as well as the ability to evaluate large population of cells in a shorter time, thus providing ac...
Inhibition of caspase-9 reduces chondrocyte apoptosis and proteoglycan loss following mechanical trauma. Chondrocyte death, a notable feature of osteoarthritis, may play a role in the initiation of cartilage degeneration. The present study was aimed at uncovering the nature and involvement of cell death in the initiation of cartilage degeneration induced by mechanical trauma. Methods: Articular cartilage discs obtained from healthy skeletally mature horses were subjected to a single-impact load (500 g from 50 mm) using a simple drop-tower device and cultured in vitro for 48 h. Chondrocyte death was examined using two independent methods: transmission electron microscopy and caspase-3 activity ass...
Validation of the sperm mobility assay in boars and stallions. The sperm mobility assay used in the present study measures the rate of sperm penetration in a biologically inert cell-separation solution (Accudenz). When a sample of sperm is overlaid in a cuvette containing Accudenz, sperm penetrate the solution and absorbance of the sample can be measured with a spectrophotometer. This assay has been successfully used to select chicken and turkey semen donors. We validated this assay for semen from boars and stallions. Absorbance was measured after overlaying fresh semen from each species in prefilled cuvettes for 1, 5, 10, 15, 20, and 40 min. There were n...
Validation of an in vitro single-impact load model of the initiation of osteoarthritis-like changes in articular cartilage. The objective of this study was the development and characterization of an in vitro model of the initiation of traumatic osteoarthritis (OA). Articular cartilage was obtained from seven healthy horses and from four horses diagnosed with OA. Cartilage disks were subjected to a single-impact load (500 g from 25, 50, or 100 mm) using a simple drop-tower device and cultured in vitro for up to 20 days. Cartilage sections were examined histologically to observe surface damage and proteoglycan loss. Percentage cell death was determined using TUNEL, release of glycosaminoglycans (GAG) to the medium wa...
Effect of maturation stage at cryopreservation on post-thaw cytoskeleton quality and fertilizability of equine oocytes. Oocyte cryopreservation is a potentially valuable technique for salvaging the germ-line when a valuable mare dies, but facilities for in vitro embryo production or oocyte transfer are not immediately available. This study examined the influence of maturation stage and freezing technique on the cryopreservability of equine oocytes. Cumulus oocyte complexes were frozen at the immature stage (GV) or after maturation in vitro for 30 hr (MII), using either conventional slow freezing (CF) or open pulled straw vitrification (OPS); cryoprotectant-exposed and untreated nonfrozen oocytes served as contr...
Equine CCL11 induces eosinophil cytoskeletal reorganization and activation. To assess the biological effects of purified recombinant equine CCL11 on equine eosinophil function. Methods: Following stimulation of eosinophils from normal horses, the polymerised form of actin was measured by flow cytometry using fluorescently labelled phalloidin. Migration was determined in a 96 well plate chemotaxis assay using 8 microm pore membranes, and adherence of eosinophils to serum-coated plastic was assessed using a colorimetric assay for eosinophil peroxidase. Superoxide generation was measured by the reduction of cytochrome C in a colorimetric assay. Results: Equine CCL11 indu...
Motility and plasma membrane integrity of spermatozoa in fractionated stallion ejaculates after storage. With the aim of investigating properties of stallion seminal plasma to eventually improve semen-handling techniques, sperm motility and plasma membrane integrity were analysed in different fractions of the ejaculates after storage. Semen was collected using a computer-controlled automated phantom that separates the ejaculates into five successive cups. Samples containing seminal plasma and skim milk extender were compared with samples stored in skim milk extender after the removal of seminal plasma by centrifugation. Fractionated ejaculates were stored cooled for 24 h after dilution with exten...
Effects of radial shock waves on membrane permeability and viability of chondrocytes and structure of articular cartilage in equine cartilage explants. To investigate in vitro effects of radial shock waves on membrane permeability, viability, and structure of chondrocytes and articular cartilage. Methods: Cartilage explants obtained from the third metacarpal and metatarsal bones of 6 horses. Methods: Equine cartilage was subjected to radial shock waves and then maintained as explants in culture for 48 hours. Treatment groups consisted of a negative control group; application of 500, 2,000, and 4,000 impulses by use of a convex handpiece (group A); and application of 500, 2,000, and 4,000 impulses by use of a concave handpiece (group B). Effec...
Effects of the omega-3 fatty acid, alpha-linolenic acid, on lipopolysaccharide-challenged synovial explants from horses. To determine the effects of pretreatment with alpha-linolenic acid, an omega-3 polyunsaturated fatty acid, on equine synovial explants challenged with lipopolysaccharide (LPS). Methods: 8 mature mixed-breed horses (4 mares and 4 geldings). Methods: Synovial explants were assigned to receive 1 of 7 concentrations of alpha-linolenic acid, ranging from 0 to 300 microg/mL. At each concentration, half of the explants were controls and half were challenged with 0.003 microg of LPS as a model of synovial inflammation. Cell inflammatory response was evaluated by measurement of prostaglandin E2 product...
Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival. Cryopreservation induces partially irreversible damage to equine sperm membranes. Part of this damage occurs due to membrane alterations induced by the membrane changing from the fluid to the gel-state as the temperature is reduced lower than the membrane transition temperature. One way to prevent this damage is to increase the membrane fluidity at low temperatures by adding cholesterol to the membrane. Different concentrations of cholesterol-loaded-cyclodextrins (CLC) were added to stallion sperm to determine the CLC concentration that optimizes cryosurvival. Higher percentages of motile sper...
Integration of future biotechnologies into the equine industry. 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...
Effect of caspase inhibitors on the post-thaw motility, and integrity of acrosome and plasma membrane of cryopreserved equine spermatozoa. The present study was designed to test the hypothesis that addition of anticaspase cocktails (inhibiting caspases and thus blocking apoptosis) to the extenders increases the post-thaw viability of equine spermatozoa. The addition of caspase inhibitors failed to improve the acrosome and plasma membrane integrity of spermatozoa, suggesting that in equine sperm cryopreservation protocols, the addition of these caspase inhibitors to cryopreservation medium may not be beneficial in protecting the sperm from the stress of cryopreservation.
Lipid peroxide formation in relation to membrane stability of fresh and frozen thawed stallion spermatozoa. In this study we used a new method to detect reactive oxygen species (ROS) induced damage at the level of the sperm plasma membrane in fresh and frozen-thawed stallion sperm. Lipid peroxidation (LPO) in sperm cells was assessed by a fluorescent assay involving the labeling of stallion sperm with the LPO reporter probe C11-BODIPY(581/591). The peroxidation dependent spectral emission shift of this membrane probe could be localized using inverted spectral confocal microscopy and quantified on living and deteriorated sperm cells using flow cytometry. Mass spectrometric analysis of the main endoge...
Effect of cryopreservation on the cellular integrity of equine embryos. Horse embryos are rarely cryopreserved in practice because expanded blastocysts tolerate freezing poorly, and the embryo begins expanding very soon after entering the uterine cavity. This study examined the effects of freezing on cytoskeleton integrity, and investigated whether cell damage could be reduced using trypsin to thin the blastocyst capsule or cytochalasin-B (cyto-B) to stabilise the cytoskeleton. Sixty-nine embryos were recovered 7 days after ovulation and equilibrated in 10% glycerol, with or without pretreatment with 0.2% trypsin or 7.5 microg/ml cyto-B. Forty-two of the embryos w...
Effect of seminal plasma on the cryopreservation of equine spermatozoa. Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80%...
In vitro comparisons of two cryopreservation techniques for equine embryos: slow-cooling and open pulled straw (OPS) vitrification. 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...
