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.
Buss DG, Giuliano E, Sharma A, Mohan RR.To determine if hybrid adeno-associated virus serotype 2/5 (AAV5) vector can effectively deliver foreign genes into the equine cornea without causing adverse side effects. The aims of this study were to: (i) evaluate efficacy of AAV5 to deliver therapeutic genes into equine corneal fibroblasts (ECFs) using enhanced green fluorescent protein (EGFP) marker gene, and (ii) establish the safety of AAV5 vector for equine corneal gene therapy. Methods: Primary ECF cultures were harvested from healthy donor equine corneas. Cultures were maintained at 37°C in humidified atmosphere with 5% CO(2). Metho...
Choi YH, Gustafson-Seabury A, Velez IC, Hartman DL, Bliss S, Riera FL, Roldán JE, Chowdhary B, Hinrichs K.The equine embryo possesses a capsule that is considered essential for its survival. We assessed viability after breaching the capsule of early (Day 6) and expanded (Day 7 and 8) equine blastocysts by micromanipulation. The capsule was penetrated using a Piezo drill, and trophoblast biopsy samples were obtained for genetic analysis. Pregnancy rates for Day-6 embryos, which had intact zonae pellucidae at the time of recovery, were 3/3 for those biopsied immediately after recovery and 2/3 for those biopsied after being shipped overnight under warm (∼28 °C) conditions. The pregnancy rates for ...
Vukojević V, Bowen AM, Wilhelm K, Ming Y, Ce Z, Schleucher J, Hore PJ, Terenius L, Morozova-Roche LA.Recent evidence supports the idea that early aggregates, protein, and lipoprotein oligomers but not large aggregates like fibrils that are formed at late stages of the aggregation process are responsible for cytotoxicity. Oligomers can interact with the cellular plasma membrane affecting its structure and/or dynamics or may be taken up by the cells. In either case, disparate cascades of molecular interactions are activated in the attempt to counteract the disturbance induced by the oligomers. If unsuccessful, cell death follows. Here, we study the molecular and cellular mechanisms underlying P...
Bourzac C, Smith LC, Vincent P, Beauchamp G, Lavoie JP, Laverty S.There is a need to assess and standardise equine bone marrow (BM) mesenchymal stem cell (MSC) isolation protocols in order to permit valid comparisons between therapeutic trials at different sites. Objective: To compare 3 protocols of equine BM MSC isolation: adherence to a plastic culture dish (Classic) and 2 gradient density separation protocols (Percoll and Ficoll). Methods: BM aspirates were harvested from the sternum of 6 mares and MSCs isolated by all 3 protocols. The cell viability after isolation, MSC yield, number of MSCs attained after 14 days of culture and the functional characteri...
Galvao AM, Ramilo DW, Skarzynski DJ, Lukasik K, Tramontano A, Mollo A, Mateus LM, Ferreira-Dias GM.Proapoptotic factor Fas ligand (FASL) and its cell surface receptor FAS are tumor necrosis factor superfamily members that trigger apoptosis in different cell types. However, their influence on luteal steroidogenesis is not clearly understood. The aim of the present work was to determine (i) the presence of the cytokine FASL and its receptor FAS in the mare's corpus luteum (CL) throughout the luteal phase, as well as (ii) the influence of FASL alone, or together with the cytokines tumor necrosis factor alpha (TNF) and interferon gamma (IFNG), on equine luteal cell production of luteotrophic an...
Buss DG, Sharma A, Giuliano EA, Mohan RR.Mitomycin C (MMC) is used clinically to treat corneal scarring in human patients. We investigated the safety and efficacy of MMC to treat corneal scarring in horses by examining its effects at the early and late stages of disease using an in vitro model. Methods: An in vitro model of equine corneal fibroblast (ECF) developed was used. The ECF or myofibroblast cultures were produced by growing primary ECF in the presence or absence of transforming growth factor beta-1 (TGFbeta1) under serum-free conditions. The MMC dose for the equine cornea was defined with dose-dependent trypan blue exclusion...
Minervini F, Lacalandra GM, Filannino A, Garbetta A, Nicassio M, Dell'aquila ME, Visconti A.Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm mo...
Jackson RE, Bormann CL, Hassun PA, Rocha AM, Motta EL, Serafini PC, Smith GD.To determine the effect of semen storage and separation techniques on sperm DNA fragmentation. Methods: Controlled clinical study. Methods: An assisted reproductive technology laboratory. Methods: Thirty normoozospermic semen samples obtained from patients undergoing infertility evaluation. Methods: One aliquot from each sample was immediately prepared (control) for the sperm chromatin dispersion assay (SCD). Aliquots used to assess storage techniques were treated in the following ways: snap frozen by liquid nitrogen immersion, slow frozen with Tris-yolk buffer and glycerol, kept on ice for 24...
Thomas CM, Whittles CE, Fuller CJ, Sharif M.To investigate whether there are any variations in chondrocyte susceptibility to an apoptotic stimulus between cells of articular cartilage (AC) from equine joints that differ in prevalence of osteoarthritis (OA). Methods: Cartilage from macroscopically normal equine metacarpophalangeal (MCP), proximal interphalangeal (PIP) and distal interphalangeal (DIP) joints was used. Prior to culture, chondrocyte viability was assessed using the fluorescein diacetate (FDA) and propidium iodide paravital staining method. AC explants were subsequently treated with tumour necrosis factor-α (TNF-α) in comb...
Vasconcelos AB, Santana MA, Santos AM, Santoro MM, Lagares MA.Microscopy has been used in the routine evaluation of sperm metabolism. Nevertheless, it has limited capacity to preview male fertility. As calorimetry may be used to evaluate directly the metabolic activity of a biological system, the aim of this study was to use microcalorimetry as an additive method for sperm metabolism evaluation of cooled equine semen. Two ejaculates of four stallions were collected and motility, viability (eosin 3%) and membrane functional integrity (hyposmotic swelling test) of spermatozoa were evaluated. Sperm samples were processed following different protocols and th...
Morrell JM, Rodriguez-Martinez H, Johannisson A.This study compared the effect on semen quality of different handling methods used in the preparation of stallion semen doses for artificial insemination. The three methods were (i) extending the ejaculate to 30-50 x 10(6)/ml, (ii) single layer centrifugation (SLC) and (iii) sperm washing (centrifugation without a colloid). An additional treatment was to add seminal plasma (SP) in various proportions to some SLC preparations. The resulting samples were evaluated for sperm motility by computer assisted sperm analysis, membrane integrity using the Nucleocounter SP-100 and chromatin integrity by ...
Lagares MA, Castanheira PN, Amaral DC, Vasconcelos AB, Veado JC, Arantes RM, Stahlberg R.The aim of the present study was to evaluate the in vitro viability of equine embryos vitrified in three different solutions. Day 6 and 6.5 embryos were measured and morphologically evaluated. Only grade 1 or 2 morulae and early blastocysts were vitrified. Eighteen embryos were distributed in Group 1: 40 percent ethylene glycol in PBS, Group: 2 and 3: 40 percent ethylene glycol, 18 percent Ficoll, 0.3M sucrose or 0.3M trehalose in PBS, respectively. The vitrified embryos were loaded individually into 0.25 ml straws, which were cooled and immersed in liquid nitrogen. After warming at 20 degree ...
Hoogewijs M, Rijsselaere T, De Vliegher S, Vanhaesebrouck E, De Schauwer C, Govaere J, Thys M, Hoflack G, Van Soom A, de Kruif A.Three experiments were conducted to evaluate the impact of centrifugation on cooled and frozen preservation of equine semen. A standard centrifugation protocol (600 x g for 10 min=CP1) was compared to four protocols with increasing g-force and decreased time period (600 x g, 1200 x g, 1800 x g and 2400 x g for 5 min for CP2, 3, 4, and 5, respectively) and to an uncentrifuged negative control. In experiment 1, the influence of the different CPs on sperm loss was evaluated by calculating the total number of sperm cells in 90% of the supernatant. Moreover, the effect on semen quality following ce...
Gamboa S, Rodrigues AS, Henriques L, Batista C, Ramalho-Santos J.A group of stallions with different reproductive indexes were used to study seasonal variations in sperm quality (Equus caballus). Semen samples were collected from late September to July and analyzed according to four seasonal periods: late September-December, January-March, late March-May, and June-July. Parameters monitored included sperm concentration, sperm motility, sperm morphology, sperm viability, acrosomal status, plasma membrane stability, and sperm mitochondrial membrane potential. Overall, seminal parameters monitored are affected mostly by time period, followed by animal and last...
Murray SJ, Santangelo KS, Bertone AL.To evaluate early cellular influences of bone morphogenetic protein (BMP)12 and BMP2 on equine superficial digital flexor tenocytes (SDFTNs) and equine bone marrow-derived mesenchymal stem cells (BMDMSCs). Methods: 9 adult clinically normal horses. Methods: BMDMSCs and SDFTNs were cultured in monolayer, either untreated or transduced with adenovirus encoding green fluorescent protein, adenovirus encoding BMP12, or adenovirus encoding BMP2. Cytomorphologic, cytochemical, immunocytochemical, and reverse transcriptase-quantitative PCR (RT-qPCR) analyses were performed on days 3 and 6. Genetic pro...
Schuh EM, Friedman MS, Carrade DD, Li J, Heeke D, Oyserman SM, Galuppo LD, Lara DJ, Walker NJ, Ferraro GL, Owens SD, Borjesson DL.OBJECTIVE-To optimize the isolation and culture of mesenchymal stem cells (MSCs) from umbilical-cord blood (UCB), identify variables that predicted successful MSC isolation, and determine whether shipping, processing, and cryopreservation altered MSC viability, recovery rates, and expansion kinetics. SAMPLE POPULATION-UCB samples from 79 Thoroughbred and Quarter Horse mares. PROCEDURES-UCB samples were processed to reduce volume and remove RBCs. Nucleated cells (NCs) were cryopreserved or grown in various culture conditions to optimize MSC monolayer expansion and proliferation. Donor and UCB-s...
Len JA, Jenkins JA, Eilts BE, Paccamonti DL, Lyle SK, Hosgood G.The objectives of this study were to determine the effects of centrifugation on equine sperm total and progressive motility, viability, and acrosomal integrity. We hypothesized that although high centrifugation forces would be detrimental to equine Equus caballus sperm, recovery rates would increase. Ejaculates from six stallions were collected, extended to a concentration of 25x10(6) cells/mL, and subjected for 10min to (1) no centrifugation (NC) or (2) centrifugation at 400xg, (3) 900xg, or (4) 4500xg. Before and after centrifugation (Day 0), and after 24h of cooling (Day 1), sperm motility ...
Laughlin AM, Welsh TH, Love CC, Varner DD, Parrish AR, Forrest DW, Ing NH.In vitro culture systems are valuable tools for investigating reproductive mechanisms in the testis. Here, we report the use of the precision-cut in vitro system using equine testicular slices. Testes were collected from immature light breed stallions (n=3) and cut into slices (mean slice weight= 13.85 ± 0.20 mg; mean slice thickness=515.00 ± 2.33 μm) using the precision-cut tissue-slicing method. Four tissue slices were placed on a grid floating on medium in individual vials. After a 1-h preincubation, they were exposed to medium containing ovine luteinizing hormone (oLH) at concentrations...
Oliveira CH, Vasconcelos AB, Souza FA, Martins-Filho OA, Silva MX, Varago FC, Lagares MA.Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen-thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 x 10(6)spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 degrees C, and cryopreserved in...
Spizziri BE, Fox MH, Bruemmer JE, Squires EL, Graham JK.Irreversible damage occurs to spermatozoal membranes, during the phase transition, when spermatozoa are cooled from room temperature to 5 degrees C. Some of this damage can be ameliorated by adding cholesterol to the membrane, thereby altering membrane lipid composition. Adding cholesterol-loaded cyclodextrins (CLCs) to stallion spermatozoa prior to freezing, increases cell cryosurvival. However, the fertilizing potential of CLC-treated stallion spermatozoa is unknown. To address this, experiments were conducted which evaluated the ability of CLC-treated stallion spermatozoa to capacitate, acr...
Glazar AI, Mullen SF, Liu J, Benson JD, Critser JK, Squires EL, Graham JK.Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cycl...
Andoh K, Kai K, Matsumura T, Maeda K.Cell lines originating from horses are necessary for isolation and propagation of equine herpesviruses (EHV). Although we established an equine-derived cell line, FHK-Tcl3, propagation ceased after fewer than 40 passages. In this study, FHK-Tcl3 cell propagation continued beyond 40 passages, achieving over 100 passages. FHK-Tcl3 cells were then cloned by limiting dilution at the 100th passage. Cloned cells were termed FHK-Tcl3.1. FHK-Tcl3.1 cells grew well and were propagated every 3 to 4 days by splitting 1:5. In addition, EHV-1, -2 and -4 showed a clear cytopathic effect (CPE) in FHK-Tcl3.1 ...
Gobeil PA, Yuan Z, Gault EA, Morgan IM, Campo MS, Nasir L.Equine sarcoids are skin tumours of horses caused by infection with BPV-1 or 2. Maintenance and replication of the viral genome depend upon the viral proteins E1 and E2. We examined the effects of an E2 specific siRNA on E2 and E1 viral gene expression, viral load and cell growth in BPV-1 transformed sarcoid-derived cells. Transfection with E2-siRNA caused a reduction in E2 and E1 mRNA expression as well as viral load, growth inhibition and decreased anchorage-independent growth. siRNA treated cells showed significantly higher apoptosis rates than control cells. Thus sequence specific targetin...
Wilhelm K, Darinskas A, Noppe W, Duchardt E, Mok KH, Vukojević V, Schleucher J, Morozova-Roche LA.Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self-assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid-liquid interface within an ion-exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed...
de Mattos Carvalho A, Alves AL, Golim MA, Moroz A, Hussni CA, de Oliveira PG, Deffune E.The purpose of this work was to isolate and cultivate mesenchymal stem cells (MSC) derived from equine adipose tissue and conduct cellular characterization with the following markers: CD90, CD44 and CD13. Adipose tissue collection was performed at the base of the horses' tails, followed by immediate isolation and cultivation of the MSC and posterior characterization by flow cytometry for the interspecies reaction test using mouse anti-rat CD90 monoclonal antibody (mAb), fluorescein isothiocyanate (FITC), and tests with specific mAb mouse anti-horse CD13 and mouse anti-horse CD44. The technique...
Cribb NC, Bouré LP, Hanna WJ, Akens MK, Mattson SE, Monteith GJ, Weese JS.To assess the antimicrobial elution characteristics, toxicity, and antimicrobial activity of amikacin-impregnated ferric-hyaluronate implants (AI-FeHAI) for amikacin delivery to the tarsocrural joint of horses. Methods: Experimental study. Methods: AI-FeHAI implants, equine cartilage, and synovium, and horses (n=6). Methods: In vitro study: Five AI-FeHAI were placed in saline solution with daily replacement until implant degradation. Eluent was tested for amikacin concentration and bioactivity. Synovial and cartilage explants were incubated in the presence or absence of AI-FeHAI for 72 hours a...
Kumar S, Kanwar S, Bansal V, Sehgal R.Diphtheria toxin has the capacity to block protein synthesis in cultured mammalian cells, and thus causing cell death. This capacity of diphtheria toxin was utilized for in-vitro neutralization test to determine antibody titer, using Vero cells, which have been found to be susceptible to diphtheria toxin. In the present study, a Vero cell assay was standardized and validated for potency estimation of diphtheria antitoxin serum (DATS). The results obtained by Vero cell assay were compared with in-vivo biological assay. High degree of correlation (+0.98) was found between in-vivo biological assa...
Choi YH, Hartman DL, Fissore RA, Bedford-Guaus SJ, Hinrichs K.We evaluated the effect of different activation methods on blastocyst development after equine nuclear transfer. All activation treatments were followed by incubation in 2 mM 6-dimethylaminopurine for 4 h. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 sec using a FemtoJet injection device, then treated with ionomycin. The blastocyst rate (9.8%) for 0.1-sec injection was significantly higher than that for 0.2 sec (0%) or 0.8 sec (1.4%). In Experiment 2, injection of murine PLCzeta cRNA before or after ionomycin treatment did not increase ...
Stewart AA, Barrett JG, Byron CR, Yates AC, Durgam SS, Evans RB, Stewart MC.To compare viability and biosynthetic capacities of cells isolated from equine tendon, muscle, and bone marrow grown on autogenous tendon matrix. Methods: Cells from 4 young adult horses. Methods: Cells were isolated, expanded, and cultured on autogenous cell-free tendon matrix for 7 days. Samples were analyzed for cell viability, proteoglycan synthesis, collagen synthesis, and mRNA expression of collagen type I, collagen type III, and cartilage oligomeric matrix protein (COMP). Results: Tendon- and muscle-derived cells required less time to reach confluence (approx 2 weeks) than did bone marr...
Kelly CM, Hoyer PB, Wise ME.Dispersed horse luteal cells were used to evaluate the ability of horse LH, hCG and PMSG to stimulate progesterone secretion in vitro. Morphological characterization of these cells before gonadotrophin stimulation indicated the presence of two populations of cells based on cell diameters. In luteal cells incubated as suspended cells, horse LH and hCG stimulated (P less than or equal to 0.05) progesterone production at all levels of treatment. Stimulation of progesterone secretion by hCG was greater (P less than or equal to 0.05) than by horse LH over the range of concentrations utilized. When ...
Thomas PG, Ball BA, Miller PG, Brinsko SP, Southwood L.Attachment of spermatozoa to oviductal epithelial cells (OEC) may be a prefertilization event in some species. We tested the hypothesis that spermatozoa that attach to equine OEC monolayers are a selected subpopulation of the initial inseminate, containing a higher proportion of morphologically normal, motile cells than the inseminate. Washed stallion spermatozoa were cocultured with monolayers of OEC or monolayers of Vero cells, and controls were incubated in wells coated with basement membrane extract (Matrigel [Mgel]) or in plastic (uncoated) wells. Unattached spermatozoa were removed by ri...
Betticher DC, Geiser J.1. The resistance of different mammalian red blood cells (RBCs) to hyperosmotic environments was studied. RBCs of six mammalian species were exposed to 10 increasingly hyperosmotic NaCl solutions for 24 hr at 5 degrees C. 2. The osmolality at which the amount of liberated haemoglobin reached a preset level (e.g. 3-4% of the total haemoglobin) showed a linear correlation with negative slope with RBC volume. This indicates that small RBCs are more resistant to hyperosmotic milieu than large ones. 3. A similar relation can be found from literature data when maximal urinary tonicities are plotted ...
Watson ED, Stokes CR, David JS, Bourne FJ, Ricketts SW.Intrauterine infusion of 1 per cent oyster glycogen solution was used to induce acute endometritis in four genitally normal mares. Numbers of viable neutrophils recovered in uterine washings had increased by 1 h after infusion and remained elevated for at least 72 h. There was a significant correlation between numbers of viable neutrophils and total protein concentrations and between prostaglandin (PG)F and PGE2 concentrations in washings. There was also a significant relationship between concentrations of 15-keto-13, 14-dihydro PGF2 alpha in plasma and PGF in washings. Intrauterine concentrat...
Gobeil PA, Yuan Z, Gault EA, Morgan IM, Campo MS, Nasir L.Equine sarcoids are skin tumours of horses caused by infection with BPV-1 or 2. Maintenance and replication of the viral genome depend upon the viral proteins E1 and E2. We examined the effects of an E2 specific siRNA on E2 and E1 viral gene expression, viral load and cell growth in BPV-1 transformed sarcoid-derived cells. Transfection with E2-siRNA caused a reduction in E2 and E1 mRNA expression as well as viral load, growth inhibition and decreased anchorage-independent growth. siRNA treated cells showed significantly higher apoptosis rates than control cells. Thus sequence specific targetin...
Hegre OD, Marshall S, Hickey GE.Plasma amine oxidase activities (benzylamine oxidase and spermine oxidase) were determined in the sera of a number of species of various ages. Benzylamine oxidase (BZO) activity, measured spectrophotometrically, was present in bovine, equine, and ovine species examined. Generally its activity in serum increased with the age of the animal. Spermine oxidase activity (SPO) was estimated by a bioassay of in vitro toxicity and did not necessarily correlate with BZO. Cytotoxicity in the presence of spermidine was found only in the sera of the ruminant species examined. Serum activity tended to rise ...
Suthar A, Maji C, Gopalkrishnan A, Raval SH, Kumar R, Kumar S.Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP). Currently, imidocarb dipropionate (ID) is the only available drug for treating the clinical form of EP. Serious side effects and incomplete clearance of infection is a major drawback of ID. Heat-shock proteins (Hsp) play a vital role in the life cycle of these haemoprotozoans by preventing alteration in protein conformation. These Hsp are activated during transmission of EP sporozoites from the tick vector (poikilotherm) to the natural host (homeotherm) and facilitate parasite survival. In the present stu...
Muhktar MM, Timoney JF.Streptococcus equi infection in horses is characterised by intense infiltration of lymph nodes by polymorphonuclear leucocytes (PMNs) suggesting a potent chemotactic response to the organism or its products. Equine PMNs were separated using Ficoll-Hypaque medium and used in an assay of chemotaxis under agarose to study the components of S equi involved in this response. Results showed that complement-derived chemotactic factors generated by activation of the alternative complement pathway were important in chemotactic responses to S equi. Both whole bacteria and peptidoglycan preparations were...
Kumar S, Kanwar S, Bansal V, Sehgal R.Diphtheria toxin has the capacity to block protein synthesis in cultured mammalian cells, and thus causing cell death. This capacity of diphtheria toxin was utilized for in-vitro neutralization test to determine antibody titer, using Vero cells, which have been found to be susceptible to diphtheria toxin. In the present study, a Vero cell assay was standardized and validated for potency estimation of diphtheria antitoxin serum (DATS). The results obtained by Vero cell assay were compared with in-vivo biological assay. High degree of correlation (+0.98) was found between in-vivo biological assa...
Gil MC, Ferrusola CO, Anel-López L, Ortiz-Rodriguez JM, Alvarez M, de Paz P, Anel L, Peña FJ.Spermatozoa undergo apoptotic changes during the cryopreservation process. These changes, recently termed spermptosis, resemble the cryopreservation induced delayed onset of cell death observed after thawing of somatic cells. Due to its importance in cryobiology, methods to easily identify spermptotic cells are warranted. In this study, a well-validated method for identification of spermatozoa with caspase 3 activity was compared with use of the combination of Hoechst 33342 (H-42) and ethidium homodimer (Eth-1). Live, dead and apoptotic spermatozoa assessed with each method were compared using...
Hosaka Y, Ozoe S, Kirisawa R, Ueda H, Takehana K, Yamaguchi M.The aim of this study was to clarify whether matrix metalloproteinases (MMP-2 and -9: gelatinases) and pro-inflammatory cytokines [tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta] are induced by heat in tendon tissue in vitro and to test the hypothesis that heat exposure causes tendinocytes to synthesize pro-inflammatory cytokines and that synthesis of these cytokines, in turn, leads to up-regulation of synthesis of gelatinases. Isolated tendinocytes from equine superficial digital flexor tendons were cultured and all experiments were performed on cells passaged 3 or 4 times. In t...
Nino-Fong R, McD￮ LA, Esparza Gonzalez BP, Kumar MR, Merschrod S EF, Poduska KM.The in vitro viability, osteogenic differentiation, and mineralization of four different equine mesenchymal stem cells (MSCs) from bone marrow, periosteum, muscle, and adipose tissue are compared, when they are cultured with different collagen-based scaffolds or with fibrin glue. The results indicate that bone marrow cells are the best source of MSCs for osteogenic differentiation, and that an electrochemically aggregated collagen gives the highest cell viability and best osteogenic differentiation among the four kinds of scaffolds studied.
Watson RE, Larson KA.Indirect immunofluorescence and lymphocyte cytotoxicity experiments demonstrated the presence of a tumor-specific antigen(s) on the surface of cells from an equine sarcoid cell line (Mc1). Autologous serum (taken from the horse from which the Mc1 cells were derived) and sera from three other sarcoid-bearing horses revealed a similar membrane immunofluorescence when reacted with Mc1 cells, indicating the existence of cross-reacting antibodies. Results of serum colony inhibition experiments indicate that these antibodies are not cytotoxic. Incubation of Mc1 cells with autologous lymphocytes resu...
Bourebaba L, Bourebaba N, Galuppo L, Marycz K.Nowadays, regenerative medicine techniques are usually based on the application of mesenchymal stem cells (MSCs) for the repair or restoration of injured damaged tissues. However, the effectiveness of autologous therapy is limited as therapeutic potential of MSCs declines due to patient's age, health condition and prolonged in vitro cultivation as a result of decreased growth rate. For that reason, there is an urgent need to develop strategies enabling the in vitro rejuvenation of MSCs prior transplantation in order to enhance their in vivo therapeutic efficiency. In presented study, we attemp...
Sergazy S, Zhetkenev S, Shulgau Z, Chulenbayeva L, Kamyshanskiy Y, Nurgaziyev M, Nurgozhina A, Mukhanbetzhanova Z, Berikkhanova K, Gulyayev A....Exosomes are cell-derived, membrane-surrounded particles that deliver bioactive molecules to various cells. Due to their small size, low immunogenicity, extended blood circulation, and involvement in cellular communication, they hold potential as effective drug carriers. Exosomes are present in various biological fluids, including mare's milk, a traditional drink in Central Asia. This study aims to compare exosome isolation methodologies and determine the stability of mare's milk-derived exosomes as potential therapeutic carriers. Three extraction methods-immunoprecipitation, size exclusion ch...
Lamar CH, Turek JJ, Bottoms GD, Fessler JF.Certain in vitro culture conditions were determined for equine endothelial cells obtained from the aorta and pulmonary arteries. Cells were enzymatically isolated from the vessel lumen, using clostridial collagenase (2.5 mg/ml of Hanks's balanced salt solution) incubated at 37 C for 30 minutes. Cells were cultured in alpha minimum essential medium supplemented with plasma-derived and nonplasma-derived bovine fetal sera, endothelial cell-growth supplement, heparin, and antibiotics. Smooth muscle cell growth was not inhibited with nonplasma-derived animal sera, plasma-derived equine serum, or he...
Kuypers FA, Easton EW, van den Hoven R, Wensing T, Roelofsen B, op den Kamp JA, van Deenen LL.The phospholipid composition and the distribution of phospholipids over the two leaflets of the membrane have been investigated for rabbit and horse erythrocyte membranes. Phosphatidylcholine (PC) comprises 39.4% and 41.3% of the total phospholipid complement of the rabbit and horse erythrocytes, respectively. In both membranes the distribution of this phospholipid is asymmetric: 70% of the PC is present in the outer layer of the rabbit membrane and 60% in that of the horse. The major species of this phospholipid class are the (1-palmitoyl-2-oleoyl)- and the (1-palmitoyl-2-linoleoyl)PC. The di...
Oldenhof H, Blässe AK, Wolkers WF, Bollwein H, Sieme H.The aim of this study was to determine the osmotic tolerance limits of stallion sperm as well as the osmotic behavior of different sperm subpopulations, including viable and non-viable cells as well as viable cells of different average sizes. A flow cytometric approach was used for simultaneous assessment of cell volume and permeability of the plasma membrane for the fluorescent dye propidium iodide while exposing the cells to media with different solute concentrations. Equine spermatozoa have limited osmotic tolerance limits: exposure to hypotonic conditions below approximately 240 mOsm kg(-1...
Duan W, Lopez MJ.OBJECTIVE To evaluate effects of various concentrations of collagenase and dimethyl sulfoxide (DMSO) on yield of equine adipose-derived multipotent stromal cells (ASCs) before and after cryopreservation. SAMPLE Supragluteal subcutaneous adipose tissue from 7 Thoroughbreds. PROCEDURES Tissues were incubated with digests containing 0.1%, 0.05%, or 0.025% type I collagenase. Part of each resulting stromal vascular fraction was cryopreserved in 80% fetal bovine serum (FBS), 10% DMSO, and 10% Dulbecco modified Eagle medium F-12 and in 95% FBS and 5% DMSO. Half of each fresh and cryopreserved hetero...
Sullivan SN, Cole SL, Stewart MC, Brokken MT, Durgam S.To investigate the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on the viability of resident cells within the fibrocartilage on the dorsal surface of the deep digital flexor tendon (FC-DDFT) and fibrocartilage on the flexor surface of the navicular bone (FC-NB) of horses. Methods: 12 to 14 explants of FC-DDFT and of FC-NB from grossly normal forelimbs of 5 cadavers of horses aged 9 to 15 years without evidence of musculoskeletal disease. Methods: Explants were incubated with culture medium (control) or TA-supplemented (0.6 or 6 mg/mL) or MPA-supplemented (0.5 or...
Verger C.We have shown previously that hemoglobin greatly stimulates chick embryo cell proliferation in Eagle's minimal essential medium supplemented with horse serum. In the present study we compared the effects of horse serum plus 10 micrometers hemoglobin to those of fetal bovine serum on subcultures of chick embryo cells serially propagated at high cell densities. The cells became elongated in the presence of fetal bovine serum and their rate of proliferation progressively decreased, whereas they became polygonal in the presence of horse serum plus hemoglobin and proliferated well in successive cel...
Papas M, Catalan J, Bonilla-Correal S, Gacem S, Miró J, Yeste M.The aim of this study was to evaluate the response of donkey spermatozoa to oxidative stress induced by hydrogen peroxide, and to determine whether the presence of seminal plasma modulates the sperm response to that stress. Nine ejaculates were collected, extended in skim milk extender and split into two aliquots. Seminal plasma was removed from the first but not second aliquot. Samples were subsequently split into four aliquots supplemented with different concentrations of commercial hydrogen peroxide (0, 100 and 250µM and 50mM). Aliquots were incubated at 37°C under aerobic conditions and ...
Chen W, Zhang Y, Li X, Chen H, Sun J, Feng F.We took advantage of gasotransmitter HS as a chemical reaction-based trigger for controlled release of doxorubicin which is precoordinated by copper ions and enclosed in horse spleen apoferritin. The nanocomposite is stable at physiological pH and temperature before HS activation. The drug release process avoids disassembly of protein shells and is controllable by the strong affinity of sulfide with copper ions. The in vitro cytotoxicity assay indicates the antitumor effect of doxorubicin toward tumor cells could be achievable by HS activation.
J Lacy K, Parlane NA, Riley CB, Gee EK, Roberts JM, McIlwraith CW.CellTrace Violet™ is a commonly used fluorescent dye used with flow cytometry to identify cell proliferation. Activated equine lymphocytes were examined using flow cytometry, microscopy and tritiated thymidine proliferation assays. CellTrace Violet™ was incorporated into the equine lymphocytes effectively. Equine lymphocytes proliferated when activated with pokeweed mitogen, but did not proliferate when previously stained with CellTrace Violet™. Serial dilutions of CellTrace Violet™ did not eliminate the inhibition of activated lymphocytes. Equine lymphocyte viability was greater than ...
Bruyninckx WJ, Blancquaert AM.Horse mononuclear cells were separated from whole blood using neutral density gradient centrifugation on Isopaque-Ficoll. The resulting cell suspension was comparable in composition with similarly prepared human and bovine mononuclear cell preparations. The relative concentration of monocytes was increased by the use of a gradient with density lower than that originally proposed by Böyum (Böyum, A. 1968. Scand. J. Clin. Lab. Investig. 21 supple. 97:77-89). Contamination by neutrophils was limited either by using a gradient medium of lower density or by replacing Isopaque-Ficoll by Percoll-0....
Du Cheyne C, Chen Y, De Craene J, Thas O, De Spiegelaere W.Accurate tools to measure RNA integrity are essential to obtain reliable gene expression data. The reverse transcription quantitative PCR (RT-qPCR) based 3':5' assay permits a direct determination of messenger RNA (mRNA) integrity. However, the use of standard curves and the possible effect of PCR inhibitors make this method cumbersome and prone to variation, especially in small samples. Here we developed a triplex digital PCR (dPCR) 3':5' assay for assessing RNA integrity in equine samples as rapid and simple alternative to RT-qPCR. This dPCR assay not only provides a straight forward analysi...
Vizcarra JA, Ford JJ.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...
