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.
Gibb Z, Lambourne SR, Quadrelli J, Smith ND, Aitken RJ.The spermatozoa of many stallions do not tolerate being cooled, restricting the commercial viability of these animals and necessitating the development of a chemically defined room temperature (RT) storage medium. This study examined the impact of two major modulators of oxidative phosphorylation, pyruvate (Pyr) and L-carnitine (L-C), on the storage of stallion spermatozoa at RT. Optimal concentrations of Pyr (10 mM) and L-C (50 mM) were first identified and these concentrations were then used to investigate the effects of these compounds on sperm functionality and oxidative stress at RT. Mito...
Leemans B, Gadella BM, Stout TA, Nelis H, Hoogewijs M, Van Soom A.Induction of hyperactivated motility is considered essential for triggering the release of oviduct-bound mammalian spermatozoa in preparation for fertilization. In this study, oviduct-bound stallion spermatozoa were exposed for 2 h to: i) pre-ovulatory and ii) post-ovulatory oviductal fluid; iii) 100% and iv) 10% follicular fluid (FF); v) cumulus cells, vi) mature equine oocytes, vii) capacitating and viii) non-capacitating medium. None of these triggered sperm release or hyperactivated motility. Interestingly, native FF was detrimental to sperm viability, an effect that was negated by heat in...
Natenstedt J, Kok AC, Dankelman J, Tuijthof GJ.Articular cartilage has limited regeneration capacities. One of the factors that appear to affect the in vitro cultivation of articular cartilage is mechanical stimulation. So far, no combination of parameters has been identified that offers the best results. The goal is to review the literature in search of the best available set of quantitative mechanical stimuli that lead to optimal in vitro cultivation.The databases Scopus and PubMed were used to survey the literature, and strict in- and exclusion criteria were applied regarding the presence of quantitative data. The review was performed b...
Leemans B, Gadella BM, Stout TA, Heras S, Smits K, Ferrer-Buitrago M, Claes E, Heindryckx B, De Vos WH, Nelis H, Hoogewijs M, Van Soom A.Coincubating equine gametes in the presence of procaine has been reported to facilitate in vitro fertilization, with cleavage rates exceeding 60%. We report that while procaine does trigger sperm hyperactivation, it independently induces cleavage of equine oocytes. First, we found that procaine (1-5 mM) did not facilitate stallion sperm penetration of equine oocytes but instead induced sperm-independent oocyte cytokinesis in the absence of the second polar body extrusion. Indeed, 56 ± 4% of oocytes cleaved within 2.5 days of exposure to 2.5 mM procaine regardless of sperm presence. However, t...
Peña FJ, Plaza Davila M, Ball BA, Squires EL, Martin Muñoz P, Ortega Ferrusola C, Balao da Silva C.The traditional assessment of stallion sperm comprises evaluation of sperm motility and membrane integrity and identification of abnormal morphology of the spermatozoa. More recently, the progressive introduction of flow cytometry is increasing the number of tests available. However, compared with other sperm structures and functions, the evaluation of mitochondria has received less attention in stallion andrology. Recent research indicates that sperm mitochondria are key structures in sperm function suffering major changes during biotechnological procedures such as cryopreservation. In this p...
Mundy LN, Ishihara A, Wellman ML, Bertone AL.To assess efficiency of gravity filtration to enhance recovery of equine bone marrow elements including stem and progenitor cells. Methods: 12 healthy adult horses. Methods: Bone marrow aspirates were collected from the fifth sternebral body and filtered by gravitational flow to obtain bone marrow elements. Raw and harvested bone marrow and marrow effluent were evaluated for WBC and platelet counts, automated and cytomorphologic cell differential counts, mesenchymal stem cell CFUs, cell viability, and differentiation capacity. Isolated cells were analyzed for CD90 and major histocompatibility ...
Gomes RG, Lisboa LA, Silva CB, Max MC, Marino PC, Oliveira RL, González SM, Barreiros TR, Marinho LS, Seneda MM.The aim of this study was to evaluate the effects of different concentrations of ascorbic acid (25, 50, and 100 μg/mL) in supplemented minimum essential medium (MEM+) on the development of equine preantral follicles that were cultured in vitro for 2 or 6 days. The contralateral ovaries (n = 5) from five mares in seasonal anestrus were collected from a local abattoir. Nine ovarian tissue fragments of approximately 5 × 5 × 1 mm were obtained from each animal. One fragment was immediately fixed and subjected to histologic analysis (control group; Day 0), and the other eight were placed in PBS ...
Collins JA, Moots RJ, Clegg PD, Milner PI.Mature articular cartilage is an avascular tissue characterized by a low oxygen environment. In joint disease, acidosis and further reductions in oxygen levels occur, compromising cartilage integrity.This study investigated how acidosis and very low oxygen levels affect components of the cellular redox system in equine articular chondrocytesand whether the antioxidants resveratrol and N-acetylcysteine could modulate this system. We used articular chondrocytes isolated from nondiseased equine joints and cultured them in a 3-D alginate bead system for 48h in <1, 2, 5, and 21% O2 at pH 7.2 or ...
Russell KA, Koch TG.Multipotent mesenchymal stromal cells (MSC) are often culture-expanded in vitro. Presently, expansion medium (EM) for MSC is supplemented with fetal bovine serum (FBS). However, increasing cost, variable composition and potential risks associated with bovine antigens call for alternatives. Platelet lysate (PL) has shown promise as an alternative supplement. Objective: To determine how equine umbilical cord blood (CB) MSC proliferate in EM enriched with PL or FBS at various concentrations. Methods: Randomised dose escalation study. Methods: Platelet concentrate was generated from 5 equine whol...
Nicpoń J, Siudzińska A, Marędziak M, Śmieszek A, Basińska K, Koszykowska M.The influences of NSAIDs (Nonsteroidal Anti-inflammatory Drugs)--non-selective metamizole and selectively-acting tolfenamic acid were estimated on morphology, ultrastructure, and cytophysiological activity of canine (Ca) and equine (Eq) adipose-derived mesenchymal stem cells (ASCs). The lowest concentration of metamizole (0.01 mg/mL) stimulated the viability and cytophysiological activity of Ca ASCs and did not affect cell morphology. Stimulated cells possessed a proper, fibroblastic shape, with large, eccentrically located nuclei. Similar effects to those observed in Ca ASCs were found in Eq ...
Moraes EA, Matos WC, Graham JK, Ferrari WD.This study was to compare the effect of adding cholesterol or cholestanol loaded cyclodextrins in stallion sperm prior to cryopreservation to optimize sperm cryosurvival. Ejaculates from each of eight stallions were diluted to 120 million cells in a S-MEDIUM diluent. The diluted sperm were sub-divided into three treatments: no additive (control); 0.75mg of cyclodextrin pre-loaded with cholesterol (CLC)/120 million sperm (positive control); 1.5mg CLC/120 million sperm; 0.75mg of cyclodextrin pre-loaded with cholestanol (CnLC)/120 million sperm; and 1.5mg CnLC/120 million sperm. To set the exper...
Wang J, Li C, Xue J, Yang J, Zhang Q, Zhang H, Chen Y.Lactobacillus helveticus isolate H9 demonstrated high angiotensin I-converting enzyme (ACE)-inhibitory activity in previous research. Here, we evaluated the fermentation characteristics (pH, titratable acidity, free amino nitrogen, and viable bacterial counts), ACE-inhibitory activity, and contents of Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) peptides of stored yogurt (4°C for 28 d) fermented by L. helveticus isolate H9 (initially inoculated at 4 concentrations), from cow, mare, and soy milks. During storage, the pH and titratable acidity remained stable in yogurts produced from all milk types ...
Radtke CL, Nino-Fong R, Rodriguez-Lecompte JC, Esparza Gonzalez BP, Stryhn H, McD￮ LA.The objectives of this study were to use non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs), to sort equine muscle tissue-derived mesenchymal stem cells (MMSCs) and bone marrow-derived mesenchymal stem cells (BMSC) into subpopulations and to carry out assays in order to compare their osteogenic capabilities. Cells from 1 young adult horse were isolated from left semitendinosus muscle tissue and from bone marrow aspirates of the fourth and fifth sternebrae. Aliquots of 800 × 10(3) MSCs from each tissue source were s...
Mageed M, Ionita C, Kissich C, Brehm W, Winter K, Ionita JC.To determine the influence of cryopreservation at two different temperatures on platelet concentration, growth factor (GF) levels and platelet activation parameters in equine ACP®; moreover, to determine if adding mechanical ACP® stimulation to freeze-thaw activation amplifies GF release from platelets. Methods: Firstly, blood from five horses was used to prepare ACP®. Platelet, platelet derived growth factor BB (PDGF-BB) and transforming growth factor β1 (TGF-β1) concentrations as well as mean platelet volume (MPV) and mean platelet component (MPC) were determined in fresh and correspond...
Drbalova J, Musilova P, Kubickova S, Sebestova H, Vahala J, Rubes J.The T cell receptor (TCR) genes (TRA, TRB, TRD and TRG) reside in 3 different chromosomal regions. During the maturation of T lymphocytes, the TCR genes are rearranged by site-specific recombination, a process that also predisposes T cells to aberrant rearrangements. Illegitimate recombination between the TCR genes occurs at a low level in healthy individuals, but this frequency may correlate with the risk of lymphoma. The aim of this work was to investigate interlocus recombination in equids. Illegitimate rearrangements were studied in peripheral blood lymphocytes by FISH with painting and BA...
Aitken JB, Naumovski N, Curry B, Grupen CG, Gibb Z, Aitken RJ.This study demonstrates for the first time the presence of an L-amino acid oxidase (LAAO) enzyme in equine spermatozoa that is able to generate significant amounts of reactive oxygen species (ROS) and create a state of oxidative stress. RT-PCR analysis revealed that the mRNA for this enzyme was present in the equine testis and spermatozoa, while immunocytochemical studies demonstrated that the mature LAAO protein was located in the sperm head, particularly in the acrosomal and postacrosomal domains. Experimental studies demonstrated that the aromatic amino acids (L-phenylalanine > L-tryptop...
Burton AG, Clark KC, Borjesson DL, Carrade DD, Burges J, Owens SD.Volume reduction and RBC depletion of equine bone marrow specimens are necessary processing steps for the immediate therapeutic use of bone marrow (BM)-derived mesenchymal stem cells (MSC), and for MSC expansion in culture. Objective: The purpose of the study was to evaluate the ability of the PrepaCyte-CB processing system to reduce volume, deplete RBC, and recover mononuclear cells (MNC) from equine BM specimens. Methods: One hundred and twenty mL of heparinized BM were obtained from each of 90 horses. A CBC was performed on the BM pre- and post-PrepaCyte-CB processing. Volume and RBC reduct...
Gaspar D, Spanoudes K, Holladay C, Pandit A, Zeugolis D.The last decade has seen significant developments in cell therapies, based on permanently differentiated, reprogrammed or engineered stem cells, for tendon injuries and degenerative conditions. In vitro studies assess the influence of biophysical, biochemical and biological signals on tenogenic phenotype maintenance and/or differentiation towards tenogenic lineage. However, the ideal culture environment has yet to be identified due to the lack of standardised experimental setup and readout system. Bone marrow mesenchymal stem cells and tenocytes/dermal fibroblasts appear to be the cell populat...
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 1 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 (P < 0.05) and VAI (...
Urra JA, Villaroel-Espíndola F, Covarrubias AA, Rodríguez-Gil JE, Ramírez-Reveco A, Concha II.Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility...
Smith RK, Garvican ER, Fortier LA.The horse is an attractive model for many human age-related degenerative diseases of the musculoskeletal system because it is a large animal species that both ages and exercises, and develops naturally occurring injuries with many similarities to the human counterpart. It therefore represents an ideal species to use as a 'proving ground' for new therapies, most notably regenerative medicine. Regenerative techniques using cell-based therapies for the treatment of equine musculoskeletal disease have been in use for over a decade. This review article provides a summary overview of the sources, cu...
Radtke CL, Nino-Fong R, Esparza Gonzalez BP, McD￮ LA.The objective of this study was to validate non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs) into subpopulations, for use with MSCs derived from equine muscle tissue, periosteal tissue, bone marrow, and adipose tissue. Cells were collected from 6 young, adult horses, postmortem. Cells were isolated from left semitendinosus muscle tissue, periosteal tissue from the distomedial aspect of the right tibia, bone marrow aspirates from the fourth and fifth sternebrae, and left supragluteal subcutaneous adipose tissue. Al...
Bourzac CA, Koenig JB, Link KA, Nykamp SG, Koch TG.To evaluate the efficacy and effects of labeling equine umbilical cord blood (UCB)- and bone marrow (BM)-derived multipotent mesenchymal stromal cells (MSCs) with an ultrasmall superparamagnetic iron oxide (SPIO) contrast agent and the detection of labeled MSCs by use of MRI. Methods: UCB MSCs from placental tissues of 5 foals and BM MSCs from 5 horses. Methods: UCB and BM MSC cultures were seeded in duplicate (5,000 cells/cm(2)). One duplicate was incubated with SPIO (50 μg/mL); the other was processed identically, but without SPIO. Mesenchymal stromal cells were expanded in triplicates for ...
Martino NA, Dell'Aquila ME, Filioli Uranio M, Rutigliano L, Nicassio M, Lacalandra GM, Hinrichs K.Evaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes. We evaluated these parameters in immature equine oocytes either fixed immediately (IMM) or held overnight in an Earle's/Hank's' M199-based medium in the absence of meiotic inhibitors (EH treatment), and in mature oocytes. We hypothesized that EH holding may affect mitochondrial function and that holding temperature may affect the efficiency of meiotic su...
Yeste M, Estrada E, Rocha LG, Marín H, Rodríguez-Gil JE, Miró J.Although cryopreservation of stallion spermatozoa allows long-term preservation of spermatozoa from particular stallions and facilitates international trade, it is understood to inflict damages on sperm cells that may finally reduce their fertilizing ability. In addition, individual differences are known to exist in the sperm ability to withstand freeze-thawing protocols. To date, these differences have mainly been reported on the basis of sperm motility and membrane integrity. For this reason, the present work sought to determine differences between good (good freezability ejaculates: GFE) an...
Petersen GF, Hilbert B, Trope G, Kalle W, Strappe P.Equine adipose-derived mesenchymal stem cells (EADMSC) provide a unique cell-based approach for treatment of a variety of equine musculoskeletal injuries, via regeneration of diseased or damaged tissue, or the secretion of immunomodulatory molecules. These capabilities can be further enhanced by genetic modification using lentiviral vectors, which provide a safe and efficient method of gene delivery. We investigated the suitability of lentiviral vector technology for gene delivery into EADMSC, using GFP expressing lentiviral vectors pseudotyped with the G glycoprotein from the vesicular stomat...
Braune S, Walter M, Schulze F, Lendlein A, Jung F.For in vitro studies assessing the interaction of platelets with implant materials, common and standardized protocols for the preparation of platelet rich plasma (PRP) are lacking, which may lead to non-matching results due to the diversity of applied protocols. Particularly, the aging of platelets during prolonged preparation and storage times is discussed to lead to an underestimation of the material thrombogenicity. Here, we study the influence of whole blood- and PRP-storage times on changes in platelet morphology and function. Blood from apparently healthy subjects was collected according...
Brazil TJ, Dixon PM, Haslett C, Murray J, McGorum BC.The aim of this study was to characterise constitutive apoptosis in equine peripheral blood neutrophils, including assessment of factors that potentially modulate neutrophil survival through alteration of the rate of constitutive apoptosis. Cells underwent spontaneous time-dependent constitutive apoptosis when aged in culture for up to 36 h, developing the structural and functional features of apoptosis observed in many cell types, including human neutrophils. Neutrophils undergoing apoptosis also had diminished zymosan activated serum (ZAS)-stimulated chemiluminescence, but maintained respo...
Donnelly KS, Giuliano EA, Sharm A, Mohan RR.To explore the effect of suberoylanilide hydroxamic acid (SAHA) (i) on corneal fibroblast differentiation, morphology, and viability; and (ii) on the expression levels of matrix metalloproteinases (MMPs) 2 and 9 using an in vitro model of equine corneal fibrosis. Methods: Healthy donor corneas were used to generate primary cultures of equine corneal fibroblasts. The fibroblasts were exposed to 5 ng/mL TGFβ1 to induce myofibroblast formation. The cultures were treated with either 5 μm or 10 μm SAHA for 72 h in the presence of TGFβ1. Real-time PCR and immunocytochemistry were used to determi...
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 (...
Schwinghamer R, Massolo A, Knight C, Klein C.This study evaluated equine endometrial explants following 12, 24, and 48 hr in culture. Measurement of an indicator of cell death in explant supernatant, light microscopy, and gene expression of biomarkers of endometrial function and cellular stress were used to compare the effect of six different media on explant viability and morphology. Viability of explants was assessed indirectly through measuring LDH activity in the culture supernatant. Regardless of culture medium composition, a significant increase in LDH activity was observed within 12 hr of culture, indicating occurrence of cell dam...
Byambaragchaa M, Lee SY, Kim DJ, Kang MH, Min KS.Previous studies showed that recombinant equine chorionic gonadotropin (rec-eCGβ/α) exhibits both follicle-stimulating hormone (FSH) and luteinizing hormone (LH)-like activities in rat LHR- and FSHR-expressing cells. In this study, we analyzed signal transduction by eelFSHR and eelLHR upon stimulation with rec-eCGβ/α and native eCG. The cyclic adenosine monophosphate (cAMP) stimulation in CHO-K1 cells expressing eelLHR was determined upon exposure to different doses (0-1,450 ng/mL) of rec-eCGβ/α and native eCG. The EC values of rec-eCGβ/α and native eCG were 172.4 and 786.6 ng/mL, resp...
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 ...
Merkies K, Chenier T, Plante C, Buhr MM.Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable, and subjectively assess only 100 to 200 spermatozoa per ejaculate. We collected two ejaculates from each of 4 stallions, and extended them to 50x10(6) sperm/mL in a nonfat dried milk solids glucose extender (EZ Mixin). Half the ejaculate was freeze-killed by immersing in liquid nitrogen for 10 min. Aliquots using appropriate volumes of live and freeze-killed spermatozoa provided the following ratios of live:dead spermatozoa: 100:0, 75:25, 50:50, 25:75, 0:100. We determined the viability of ea...
Garvican ER, Salavati M, Smith RKW, Dudhia J.The purpose of this study was to investigate the effect of normal synovial fluid (SF) on exposed endogenous tendon-derived cells (TDCs) and engrafted mesenchymal stem cells (MSCs) within the tendon extracellular matrix. Methods: Explants from equine superficial digital flexor (extra-synovial) and deep digital flexor tendons (DDFTs) from the compressed, intra-synovial and the tensile, extra-synovial regions were cultured in allogeneic or autologous SF-media. Human hamstring explants were cultured in allogeneic SF. Explant viability was assessed by staining. Proliferation of equine monolayer MSC...
Viganò M, Tessaro I, Trovato L, Colombini A, Scala M, Magi A, Toto A, Peretti G, de Girolamo L.The management of cartilage lesions is an open issue in clinical practice, and regenerative medicine represents a promising approach, including the use of autologous micrografts whose efficacy was already tested in different clinical settings. The aim of this study was to characterize in vitro the effect of autologous cartilage micrografts on chondrocyte viability and differentiation and perform an evaluation of their application in racehorses affected by joint diseases. Methods: Matched human chondrocytes and micrografts were obtained from articular cartilage using Rigenera® procedure. Chond...
Haag KT, Magalhães-Padilha DM, Fonseca GR, Wischral A, Gastal MO, King SS, Jones KL, Figueiredo JR, Gastal EL.The aims of this study in mares were to: (1) compare preantral follicle parameters between in vitro Biopsy Pick-Up (BPU) and scalpel blade collection methods and between histological and mechanical isolation processing (experiment 1); (2) histologically evaluate preantral follicles (experiment 2); and (3) compare histological analysis with a previously established mechanical isolation technique using a tissue chopper (experiment 3) for ovarian cortical fragments obtained in vivo using a BPU instrument. In experiment 1, preantral follicles were analyzed (N = 220; 90% primordial and 10% primary)...
Alamaary MS, Haron AW, Hiew MWH, Ali M.Present study aimed to investigate the effect of adding antioxidants, cysteine and ascorbic acid on the levels of glutamic oxaloacetic transaminase (GOT), glutamic-pyruvate (GPT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and γ-glutamyl transpeptidase (GGT) enzymes of post-thawed stallion sperm. Ten ejaculates were collected each from four healthy stallions and cryopreserved using HF-20 freezing extender containing either 0 mg/ml cysteine or ascorbic acid, 0.5 mg/ml cysteine and 0.5 mg/ml ascorbic acid. All samples in freezing extender containing cysteine or ascorbic acid or ...
Martín Muñoz P, Anel-López L, Ortiz-Rodríguez JM, Álvarez M, de Paz P, Balao da Silva C, Rodríguez Martinez H, Gil MC, Anel L, Peña FJ....Oxidative stress is a major factor explaining sperm dysfunction of spermatozoa surviving freezing and thawing and is also considered a major inducer of a special form of apoptosis, visible after thawing, in cryopreserved spermatozoa. To obtain further insights into the link between oxidative stress and the induction of apoptotic changes, stallion spermatozoa were induced to oxidative stress through redox cycling after exposure to 2-methyl-1,4-naphthoquinone (menadione), or hydroxyl radical formation after FeSO exposure. Either exposure induced significant increases (p < 0.05) in two marke...
Oldenhof H, Bigalk J, Hettel C, de Oliveira Barros L, Sydykov B, Bajcsy ÁC, Sieme H, Wolkers WF.In this study, modeling and experimental approaches were used to investigate the interplay between cooling rate and protectant concentration for cryopreservation of stallion sperm. Glycerol (GLY), ethylene glycol (EG), dimethylformamide (DMF), propylene glycol (PG), and dimethyl sulfoxide (DMSO) were tested as cryoprotective agents (CPAs), using concentrations up to 1500 mM and cooling rates ranging from 5°C to 55°C min. Modeling of the extent of sperm dehydration during freezing was done using previously determined values of the sperm membrane permeability to water to predict optimal cool...
Hinrichs K.Methods for holding of oocytes and embryos during shipment as well as for their cryopreservation can greatly aid equine reproductive management. Oocytes can be held at room temperature overnight or at cooler temperatures for two nights without affecting maturation or embryo development after intracytoplasmic sperm injection. In contrast, methods for cryopreservation of equine oocytes that support high rates of embryo development have not yet been established. Equine embryos may be held overnight at temperatures from 5°C to 19°C without reduction in viability, but longer holding periods, or h...
Martino NA, Dell'Aquila ME, Filioli Uranio M, Rutigliano L, Nicassio M, Lacalandra GM, Hinrichs K.Evaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes. We evaluated these parameters in immature equine oocytes either fixed immediately (IMM) or held overnight in an Earle's/Hank's' M199-based medium in the absence of meiotic inhibitors (EH treatment), and in mature oocytes. We hypothesized that EH holding may affect mitochondrial function and that holding temperature may affect the efficiency of meiotic su...
Linfor JJ, Meyers SA.Single-cell gel electrophoresis (SCGE), or comet assay, has the ability to detect damage at the single cell level and has not been reported for equine sperm. The ability to detect nuclear damage at the single cell level could aid in the advancement of protocols for optimal semen preservation. The goals of these experiments were to adapt this assay for use with equine sperm and to utilize the assay for determining the integrity of equine sperm DNA following treatments with storage at various decreased temperatures (-20 degrees C and 5 degrees C). Results from experiments in which sperm were fro...
Redina OE, Amstislavsky SYa , Maksimovsky LF.This study examined the developmental capacity of oocytes in DD mice after they had been injected with pregnant mares' serum gonadotrophin at different stages of the oestrous cycle. The superovulation of mature DD mice at pro-oestrus, oestrus and metoestrus resulted in a large yield of viable embryos. The proportion of abnormal embryos was highest after injection of pregnant mares' serum gonadotrophin at dioestrus. The pool of viable oocytes was most synchronized with normal development after the hormone was injected at oestrus. The results demonstrate that oocytes of different morphology coul...
Malisauskas M, Darinskas A, Zamotin VV, Gharibyan A, Kostanyan IA, Morozova-Roche LA.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...
Sasao T, Fukuda Y, Yoshida S, Miyabara S, Kasashima Y, Kuwano A, Arai K.In regenerative medicine using transplantation of mesenchymal stem cells (MSCs), the importance of regulating the quality of MSCs has been well recognized; however, there is little information concerning the relationship between the population doubling level (PDL) and the stemness of MSCs in equine medicine. In this study, we showed that the amount of glycosaminoglycan (GAG) secreted by bone marrow-derived MSCs (BMSCs) decreases with increase of PDL. Enzymatic digestion and two-dimensional electrophoresis revealed that a main component of GAG produced by BMSCs was hyaluronan with a small amoun...
Radtke CL, Nino-Fong R, Rodriguez-Lecompte JC, Esparza Gonzalez BP, Stryhn H, McD￮ LA.The objectives of this study were to use non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs), to sort equine muscle tissue-derived mesenchymal stem cells (MMSCs) and bone marrow-derived mesenchymal stem cells (BMSC) into subpopulations and to carry out assays in order to compare their osteogenic capabilities. Cells from 1 young adult horse were isolated from left semitendinosus muscle tissue and from bone marrow aspirates of the fourth and fifth sternebrae. Aliquots of 800 × 10(3) MSCs from each tissue source were s...
Al-Essawe EM, Johannisson A, Wulf M, Aurich C, Morrell JM.Freezing and thawing processes induce structural and functional damage to sperm plasma membranes and internal organelles. Adding seminal plasma (SP) has been found to minimize or repair the cryoinjuries in some species. The objective of this study was to investigate whether adding SP from stallions of known freezability after thawing could repair cryoinjuries. Semen was collected from warmblood stallions (n = 8, three ejaculates/stallion) and processed by Single Layer Centrifugation (SLC) to remove SP prior to freezing. Pooled SP (5%) from bad freezer (BF) or good freezer (GF) stallions wa...
Gomes RG, Andrade ER, Lisboa LA, Ciquini A, Barreiros TR, Fonseca NA, Seneda MM.The objective was to evaluate the efficiency of phosphate-buffered saline (PBS) and Minimum Essential Medium (MEM) during the transport of equine preantral and antral follicles at various temperatures and incubation interval. Equine ovaries (n = 10) from an abattoir were cut into 19 fragments; one was immediately fixed in Bouin's solution (control) and the other fragments were placed in PBS or MEM solution at 4, 20, or 39 °C for 4, 12, or 24 h. After the respective incubation periods, all fragments were fixed in Bouin's solution for 24 h and then submitted to standard histologic analysis. In ...
Murphy C, English AM, Holden SA, Fair S.An unacceptable proportion of stallion sperm do not survive the freeze-thaw process. The hypothesis of this study was that adding cholesterol to a stallion semen extender would stabilise the sperm membrane, resulting in an improved post-thaw semen quality in terms of increased sperm viability, membrane integrity and fluidity, and reduced oxidative stress. Semen was collected from three stallions and diluted in four extenders: TALP; TALP+0.75mg methyl-β-cyclodextrin-cholesterol (MβCD)/mL (MβCD0.75); TALP+1.5mg MβCD-cholesterol/mL (MβCD1.5); and Equipro. Following 15min incubation, samples ...
The Journal of parasitologyJanuary 23, 2003
Volume 88, Issue 6 1252-1254 doi: 10.1645/0022-3395(2002)088[1252:EOHTAD]2.0.CO;2
Dubey JP, Saville WJ, Sreekumar C, Shen SK, Lindsay OS, Pena HF, Vianna MC, Gennari SM, Reed SM.The effect of moist heat and several disinfectants on Sarcocystis neurona sporocysts was investigated. Sporocysts (4 million) were suspended in water and heated to 50, 55, 60, 65, and 70 C for various times and were then bioassayed in interferon gamma gene knockout (KO) mice. Sporocysts heated to 50 C for 60 min and 55 C for 5 min were infective to KO mice, whereas sporocysts heated to 55 C for 15 min and 60 C or more for 1 min were rendered noninfective to mice. Treatment with bleach (10, 20, and 100%), 2% chlorhexidine, 1% betadine, 5% o-benzyl-p-chlorophenol, 12.56% phenol, 6% benzyl ammoni...
Dietze B, Cierpka E, Schäfer M, Schill W, Lutz MB.Equine dendritic cells (eqDC) can be generated from peripheral blood monocytes by propagation in GM-CSF and IL-4. Despite similarities with the generation of human DC, we found significant improvements for eqDC generation and functional influences on eqDC maturation. The fractionation of peripheral blood mononuclear cells (PBMC) by two subsequent gradients at densities of 1.090 and 1.077 as well as an adherence step in AIM V((R)) medium on dishes coated with extracellular matrix components (Primaria) improved the purity and yield of DC. After 3 days, eqDC cultures with GM-CSF alone developed i...
Devireddy RV, Swanlund DJ, Alghamdi AS, Duoos LA, Troedsson MH, Bischof JC, Roberts KP.The effects of extracellular ice and cryoprotective agents on the measured volumetric shrinkage response and the membrane permeability parameters of equine spermatozoa have been reported previously. The volumetric shrinkage data were obtained using a differential scanning calorimeter technique that was independent of cell shape. The aim of this study was to examine the effects of collection and cooling conditions on the motility and the water transport parameters at subzero temperatures of equine spermatozoa. Stallion semen samples were collected using either a commercial lubricating agent, wh...
Cheng FP, Gadella BM, Voorhout WF, Fazeli A, Bevers MM, Colenbrander B.The aim of the present study was to investigate whether the induction of stallion sperm acrosome reaction (AR) by progesterone is mediated by binding of progesterone to a receptor on the sperm plasma membrane or to an intracellular progesterone receptor. Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) in combination with a vital stain, ethidium homodimer, was applied to visualize the presence of the progesterone receptor on living spermatozoa. Alternatively, an indirect immunofluorescence technique employing a monoclonal antibody (C-262) against human intracellu...
Palomino Lago E, Jelbert ER, Baird A, Lam PY, Guest DJ.Persistent inflammation is associated with the poor regeneration of musculoskeletal tissues. Embryonic stem cells (ESCs) have an attenuated response to inflammatory cytokines, but there are mixed reports on the response of induced pluripotent stem cells (iPSCs) to inflammation. Horses provide a relevant large animal model for studying musculoskeletal tissue diseases and the testing of novel therapies. The aim of this study was to determine if equine iPSCs are responsive to the inflammatory cytokines IL-1β, TNFα and IFN-γ in their undifferentiated state, or following differentiation into ten...
Weiss DJ, Kraemer R, Schmit K.A simple discontinuous Percoll density-gradient technique was adapted for isolation of granulocytes and mononuclear cells from cats, dogs, horses and cattle. Separation was accomplished at low speeds using a standard tabletop centrifuge. Cell purity was 100% for both granulocytes and mononuclear cells and cell viability exceeded 95%. Percent recovery of leukocytes ranged from 69 to 83%.
Scare JA, Dini P, Norris JK, Steuer AE, Scoggin K, Gravatte HS, Howe DK, Slusarewicz P, Nielsen MK.Ascarid parasites infect a variety of hosts and regular anthelmintic treatment is recommended for all species. Parascaris spp. is the only ascarid species with widespread anthelmintic resistance, which allows for the study of resistance mechanisms. The purpose of this study was to establish an in vitro drug exposure protocol for adult anthelmintic-naïve Parascaris spp. and report a preliminary transcriptomic analysis in response to drug exposure. Live worms were harvested from foal necropsies and maintained in RPMI-1640 at 37 °C. Serial dilutions of oxibendazole (OBZ) and ivermectin (IVM) we...
Adler DMT, Frellesen JF, Karlsen CV, Jensen LD, Dahm ASQ, Berg LC.To investigate the in vitro effects of clinically relevant concentrations of the local anesthetics (LAs) bupivacaine, lidocaine, lidocaine with preservative (LP), mepivacaine, and ropivacaine on equine chondrocyte and fibroblast-like synoviocyte (FLS) viability. Methods: Chondrocytes and FLSs of the metacarpophalangeal joints of 4 healthy adult horses. Methods: Viability of chondrocytes and FLSs was determined with 3 assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and trypan blue (TB) exclusion (only FLS). Viability was assessed after 30...
Schaufuss P, Müller F, Valentin-Weigand P.Haemolytic activities of Trichophyton (T.) mentagrophytes were detected and characterized by qualitative and quantitative assays. On Columbia agar supplemented with blood from horses, cattle or sheep, T. mentagrophytes expressed a strong zone of complete haemolysis. No haemolytic activities could be detected in the closely related T. verrucosum var. ochraceum. The same results were obtained after cultivation of the fungi on sterile cellulose acetate filters placed on the surface on Columbia blood agar. After removal of the filter, complete haemolysis was detected below the colony of T. mentagr...
