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.
Metz GE, Abeyá MM, Serena MS, Panei CJ, Echeverría MG.Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation,...
Pereira BC, Ortiz I, Dorado J, Consuegra C, Diaz-Jimenez M, Demyda-Peyras S, Gosalvez J, Hidalgo M.DNA fragmentation of granulosa cells might be related to developmental competence of the equine oocyte. Granulosa cells are commonly stored before DNA fragmentation assessment, but the effect of preservation methods on this parameter remains unexplored. The aim of this study was to evaluate whether or not cryopreservation of granulosa cells affects the DNA damage. Equine oocytes were recovered from postmortem ovaries of five mares. Granulosa cells were washed by centrifugation and then analyzed (control) or stored in cryovials following four different protocols: P1 = directly plunged in liqui...
Santos VH, Pfeifer JPH, de Souza JB, Milani BHG, de Oliveira RA, Assis MG, Deffune E, Moroz A, Alves ALG.The original article [1] contained a minor error regarding the mean diameter of the alginate microcapsules described in relation to Fig. 4 in the Results section. The microcapsules had an actual mean diameter of 3000 μm instead of 1000 μm as mistakenly mentioned in the original article.
Max MC, Bizarro-Silva C, Búfalo I, González SM, Lindquist AG, Gomes RG, Barreiros TRR, Lisboa LA, Morotti F, Seneda MM.Folliculogenesis is a process of development and maturation of the ovarian follicles, being essential for the maintenance of fertility. In in vivo conditions, 99.9% of the follicles of an ovary do not ovulate and undergo atresia. In order to minimize this loss and to clarify the existing mechanisms, a technique was developed that allows for the in vitro follicular development. The objective of this study was to evaluate the effects of different epidermal growth factor (EGF) concentrations on the in vitro culturing of equine preantral follicles. Ovaries (n = 10) were collected from a local ...
Arroyo-Salvo C, Sanhueza F, Fuentes F, Treulén F, Arias ME, Cabrera P, Silva M, Felmer R.Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capa...
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...
Kisiday JD, Colbath AC, Tangtrongsup S.Ex vivo induction of chondrogenesis is a promising approach to improve upon the use of bone marrow mesenchymal stem cells (MSCs) for cartilage tissue engineering. This study evaluated the potential to induce chondrogenesis with days of culture in chondrogenic medium for MSCs encapsulated in self-assembling peptide hydrogel. To simulate the transition from preconditioning culture to implantation, MSCs were isolated from self-assembling peptide hydrogel into an individual cell suspension. Commitment to chondrogenesis was evaluated by seeding preconditioned MSCs into agarose and culturing in the ...
Brandt L, Schubert S, Scheibe P, Brehm W, Franzen J, Gross C, Burk J.Transplantation of multipotent mesenchymal progenitor cells is a valuable option for treating tendon disease. Tenogenic differentiation leading to cell replacement and subsequent matrix modulation may contribute to the regenerative effects of these cells, but it is unclear whether this occurs in the inflammatory environment of acute tendon disease. Equine adipose-derived stromal cells (ASC) were cultured as monolayers or on decellularized tendon scaffolds in static or dynamic conditions, the latter represented by cyclic stretching. The impact of different inflammatory conditions, as represente...
Ursini TL, Amelse LL, Elkhenany HA, Odoi A, Carter-Arnold JL, Adair HS, Dhar MS.Bone marrow-derived mesenchymal stem cells (BM-MSCs) are frequently used in the treatment of musculoskeletal injuries. Fully characterised cells that are readily available for use is optimum. Allogenic BM-MSCs can satisfy the need for rapid treatment, however, their safety has been questioned. Objective: Objectives were to characterise BM-MSCs from an adult donor horse, in vitro, and to identify and describe adverse reactions that occurred following their injection into other horses. We hypothesised that BM-MSCs capable of proliferation, differentiation and lacking MHC II from one donor could ...
Barrachina L, Romero A, Zaragoza P, Rodellar C, Vázquez FJ.Since the clinical use of mesenchymal stem cells (MSCs) for treating musculoskeletal injuries is gaining popularity, practitioners should be aware of the factors that may affect MSCs from tissue harvesting for MSC isolation to cell delivery into the injury site. This review provides equine practitioners with up-to-date, practical knowledge for the treatment of equine patients using MSCs. A brief overview of laboratory procedures affecting MSCs is provided, but the main focus is on shipping conditions, routes of administration, injection methods, and which commonly used products can be combined...
Haysom LZ, Lee-Fowler TM, Spangler EA.Storage and temperature significantly impact bronchoalveolar lavage fluid (BALF) analysis, and shipment of samples to diagnostic laboratories is often necessary. Alternative sample preparation methods could limit storage and temperature effects. Objective: This study aimed to determine if airway wash samples that were fixed in formalin after being embedded in Histogel or Gelfoam gave comparable results to fresh cytocentrifuged or sediment smear preparations for the evaluation of cell morphology. Methods: Eleven bronchoalveolar lavage and 3 transtracheal wash fluids were available, including 8 ...
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...
Brandt AM, Kania JM, Gonzalez ML, Johnson SE.Hepatocyte growth factor (HGF) signals mediate mouse skeletal muscle stem cell, or satellite cell (SC), reentry into the cell cycle and myoblast proliferation. Because the athletic horse experiences exercise-induced muscle damage, the objective of the experiment was to determine the effect of HGF on equine SC (eqSC) bioactivity. Fresh isolates of adult eqSC were incubated with increasing concentrations of HGF and the initial time to DNA synthesis was measured. Media supplementation with HGF did not shorten (P > 0.05) the duration of G0/G1 transition suggesting the growth factor does not aff...
Nouri H, Shojaeian K, Jalilvand G, Kohram H.The objective was to assess the influence of pomegranate seed oil supplementation on the quality of fresh, cooled and frozen-thawed Arabian breed stallion semen. Eight stallions (n = 4 per group) received their normal diet (control group) or normal diet top dressed with 200 ml of pomegranate seed oil (PSO group). Semen was collected every fifteen days for 90 days. Stallions were reversed across the treatments after a sixty-day interval. In cooled and stored condition (2, 12 and 24 hr), spermatozoa motion characteristics, membrane integrity, viability, morphology and lipid peroxidation wer...
Bertram CA, Dietert K, Pieper L, Erickson NA, Barton AK, Klopfleisch R.During bronchoalveolar lavage fluid (BALF) sample preparation in horses, several technical aspects can affect sample variability. To date, the effects of different fixatives on prepared equine BALF films have been insufficiently investigated. Objective: This study aimed to determine the effect of various on-slide fixation methods on cell quality, including spray fixation of wet films, and acetone and methanol fixation of air-dried samples in comparison with unfixed, air-dried films. Methods: Cytocentrifuged BALF samples from 5 horses were fixed in a wet state using a commercially available fix...
Santos RCD, Pinho FA, Passos GP, Larangeira DF, Barrouin-Melo SM.The most commonly used culture medium for the in vitro isolation of Leishmania spp. from canine biological samples is biphasic Novy-MacNeal-Nicolle (NNN) medium, whose solid phase is prepared using rabbit blood. Leishmania infantum parasites from natural infections are highly sensitive and demanding for growth in axenic conditions when firstly obtained from the dog's body. The objective of this study was to evaluate whether NNN medium (NNN-test) prepared with chicken blood (NNN-C), ox blood (NNN-O), horse blood (NNN-H) or sheep blood (NNN-S) was viable for the isolation of parasites from natur...
Rotter Sopasakis V, Wickelgren R, Sukonina V, Brantsing C, Svala E, Hansson E, Enerbäck S, Lindahl A, Skiöldebrand E.Chondrocytes are responsible for remodeling and maintaining the structural and functional integrity of the cartilage extracellular matrix. Because of the absence of a vascular supply, chondrocytes survive in a relatively hypoxic environment and thus have limited regenerative capacity during conditions of cellular stress associated with inflammation and matrix degradation, such as osteoarthritis (OA). Glucose is essential to sustain chondrocyte metabolism and is a precursor for key matrix components. In this study, we investigated the importance of glucose as a fuel source for matrix repair dur...
Galvão A, Wolodko K, Rebordão MR, Skarzynski D, Ferreira-Dias G.In the present report we describe the involvement of transforming growth factor B1 (TGF) in functional regression and structural luteolysis in the mare. Firstly, TGF and its receptors activin-like kinase (ALK) 5 and TGF receptor 2 were identified in corpus luteum (CL) steroidogenic, endothelial and fibroblast-like cells. Also, TGF and ALK5 protein expression were shown to be increased in Mid-, and Late-CL (p < 0.05). Subsequently, using an in vitro model with Mid-CL cells, we studied the role of TGF on secretory activity and cell viability. Cell treatment with TGF decreased progesterone...
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...
Anderson SL, Townsend HGG, Singh B.OBJECTIVE To evaluate the effect of lipopolysaccharide (LPS) on apoptosis of equine neutrophils in vitro. SAMPLE Venous blood samples from 40 adult horses. PROCEDURES Neutrophils were isolated from blood samples and cultured with or without LPS from Escherichia coli O55:B5 for 12 or 24 hours. Neutrophil apoptosis was assessed by use of cytologic examination, annexin V and propidium iodide staining quantified with flow cytometry, coincubation with inducers of intrinsic and extrinsic apoptosis or a toll-like receptor (TLR) 4 inhibitor, and measurement of caspase-3, -8, and -9 activities. RESULTS...
Santos VH, Pfeifer JPH, de Souza JB, Milani BHG, de Oliveira RA, Assis MG, Deffune E, Moroz A, Alves ALG.Mesenchymal stem cells derived from the synovial membrane (MSCSM) have a greater potential for joint regeneration, besides the capacity for chondrogenic differentiation, since they are a source closer to the chondrocytes. This study aimed to cultivate and evaluate viability and differentiation of MSC encapsulated in a three-dimensional alginate hydrogel (HA) scaffold. Samples of the synovial membrane of the metatarsophalangeal joint of 4 horses were collected by astroscopic surgery. These were subjected to enzymatic digestion, isolated mesenchymal cells, cultured in monolayers and encapsulated...
Varela E, Rey J, Plaza E, Muñoz de Propios P, Ortiz-Rodríguez JM, Álvarez M, Anel-López L, Anel L, De Paz P, Gil MC, Morrell JM....Contaminating bacteria present in stallion ejaculates may compromise sperm quality during storage. Different procedures have been used to reduce the load of microorganisms in semen and avoid bacterial growth during storage. The aims of this study were: 1) to evaluate different techniques to eliminate bacteria in semen 2) to study the relationship between total microflora load (TML) and ROS production; and 3) to determine if TML affects the functionality of cool-stored sperm. Ejaculates from 11 stallions were split and processed in 3 ways: A. extended semen; B. conventional centrifuged semen, a...
Naskou MC, Sumner SM, Chocallo A, Kemelmakher H, Thoresen M, Copland I, Galipeau J, Peroni JF.Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) poo...
Castro Martins M, Peffers MJ, Lee K, Rubio-Martinez LM.Intra-articular administration of stanozolol has shown promising results by improving the clinical management of lameness associated with naturally-occurring osteoarthritis (OA) in horses, and by decreasing osteophyte formation and subchondral bone reaction in sheep following surgically induced OA. However, there is limited evidence on the anti-inflammatory and modulatory properties of stanozolol on articular tissues. The objective of the current study was to evaluate the effects of stanozolol on chondrocyte viability and gene expression in normal equine chondrocytes and an inflammatory in vit...
Duan W, Chen C, Haque M, Hayes D, Lopez MJ.Use of bioscaffolds to direct osteogenic differentiation of adult multipotent stromal cells (MSCs) without exogenous proteins is a contemporary approach to bone regeneration. Identification of in vivo osteogenic contributions of exogenous MSCs on bioscaffolds after long-term implantation is vital to understanding cell persistence and effect duration. This study was designed to quantify in vivo equine MSC osteogenesis on synthetic polymer scaffolds with distinct mineral combinations 9 weeks after implantation in a murine model. Cryopreserved, passage (P)1, equine bone marrow-derived MSCs (BMSC)...
Ceriotti S, Consiglio AL, Casati L, Cremonesi F, Sibilia V, Ferrucci F.Increasing evidence suggests a role for ghrelin in the control of articular inflammatory diseases like osteoarthritis (OA). In the present study we examined the ability of ghrelin to counteract LPS-induced necrosis and apoptosis of chondrocytes and the involvement of GH secretagogue receptor (GHS-R)1a in the protective action of ghrelin. The effects of ghrelin (10-10 mol/L) on equine primary cultured chondrocytes viability and necrosis in basal conditions and under LPS treatment (100 ng/ml) were detected by using both acridine orange/propidium iodide staining and annexin-5/propidium iodide...
Diaw M, Salgado RM, Canesin HS, Gridley N, Hinrichs K.Intracytoplasmic sperm injection (ICSI) is an important tool for equine embryo production in both clinical and research settings. In clinical ICSI programs, immature equine cumulus-oocyte complexes (COCs) are often collected at the mare's location and shipped to the ICSI laboratory. To simplify shipment and aid scheduling of subsequent procedures, COCs can be held overnight at room temperature (∼22 °C) before placement into maturation culture, with no detrimental effect on meiotic or developmental competence. A recent study indicated that it might be possible to hold COCs overnight at col...
Reisbig NA, Hussein HA, Pinnell E, Bertone AL.OBJECTIVE To create a bioactive synovium scaffold by infusing decellularized synovial-derived extracellular matrix (synECM) with synovial-derived mesenchymal stem cells (synMSCs). SAMPLE Synovium from the femoropatellar and medial femorotibial joints of equine cadavers. PROCEDURES The synMSCs were cultured in monolayer and not treated or cotransduced to enhance expression of green fluorescent protein (GFP) and human bone morphogenetic protein (BMP)-2. The synECM was decellularized with 0.1% peracetic acid and then seeded with synMSCs (0.5 × 10 cells/0.5 mL) by use of a 30% serum gradient. Sam...
Brom-de-Luna JG, Canesin HS, Wright G, Hinrichs K.Nuclear transfer using somatic cells from frozen semen (FzSC) would allow cloning of animals for which no other genetic material is available. Horses are one of the few species for which cloning is commercially feasible; despite this, there is no information available on the culture of equine FzSC. After preliminary trials on equine FzSC, recovered by density-gradient centrifugation, resulted in no growth, we hypothesized that sperm in the culture system negatively affected cell proliferation. Therefore, we evaluated culture of FzSC isolated using fluorescence-assisted cell sorting. In Exp. 1,...
Ishikawa S, Horinouchi C, Mizoguchi R, Senokuchi A, Kamikakimoto R, Murata D, Hatazoe T, Tozaki T, Misumi K, Hobo S.The sizes of Japanese native horses have drastically decreased, and protection of these populations is important for Japanese horse culture. Social trials as well as scientific attempts are necessary for maintaining the breed. Mesenchymal stem cells (MSCs) have potential as a cell source for various cell therapies. However, there have been no reports on MSCs of Japanese native horses. We aimed to isolate and characterize MSCs from a Japanese native horse, the Noma horse. Plastic-adherent and self-replicating cells were isolated from a Noma horse's peripheral blood (PB). The isolated cells had ...
Hagen A, Holland H, Brandt VP, Doll CU, Häußler TC, Melzer M, Moellerberndt J, Lehmann H, Burk J.Platelet lysate (PL) is an attractive platelet-based therapeutic tool and has shown promise as xeno-free replacement for fetal bovine serum (FBS) in human and equine mesenchymal stromal cell (MSC) culture. Here, we established a scalable buffy-coat-based protocol for canine PL (cPL) production (n = 12). The cPL was tested in canine adipose MSC (n = 5) culture compared to FBS. For further comparison, equine adipose MSC (n = 5) were cultured with analogous equine PL (ePL) or FBS. During canine blood processing, platelet and transforming growth factor-β1 concentrations increased ( < 0.05 and < ...
Catalán J, Yánez-Ortiz I, Tvarijonaviciute A, González-Arostegui LG, Rubio CP, Yeste M, Miró J, Barranco I.This study investigated whether the activities of the antioxidant components of donkey seminal plasma (SP)-both enzymatic (superoxide dismutase (SOD), catalase-like (CAT), glutathione peroxidase-like (GPX), and paraoxonase type 1 (PON1)) and non-enzymatic (measured in terms of total thiol, copper-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP), and Trolox equivalent antioxidant capacity (TEAC))-and oxidative stress index (OSI) are related to sperm cryotolerance. For this purpose, 15 ejaculates from jackasses (one per individual) were collected and split into tw...
Pommer AC, Rutllant J, Meyers SA.Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0.05) in anisosmolal conditio...
Delgado-Bermúdez A, Noto F, Bonilla-Correal S, Garcia-Bonavila E, Catalán J, Papas M, Bonet S, Miró J, Yeste M.Aquaporins (AQPs), a family of ubiquitous water channels divided into orthodox AQPs, aquaglyceroporins (GLPs), and superAQPs, are present in stallion spermatozoa. The aim of this study was to elucidate the functional relevance of each group of AQPs during stallion sperm cryopreservation through the use of three different inhibitors: acetazolamide (AC), phloretin (PHL) and propanediol (PDO). Sperm quality and function parameters were evaluated in the presence or absence of each inhibitor in fresh and frozen-thawed samples. In the presence of AC, different parameters were altered ( < 0.05), b...
Metcalf GL, McClure SR, Hostetter JM, Martinez RF, Wang C.Use of mesenchymal stem cells (MSCs) found in the stromal vascular fraction (SVF) of equine adipose tissue has promising applications for regenerative therapies. The most commonly used source of equine adipose tissue is the subcutaneous tailhead. The objective of this study was to compare 3 adipose depot sites in horses and determine the viability and cellular yield, capillary density, gene expression for selected markers, and colony-forming unit fibroblasts (CFU-Fs) in adipose tissue taken from these sites. Adipose tissue was excised from the area lateral to the tailhead, the inguinal region,...
Maury W, Wright PJ, Bradley S.A novel strain of equine infectious anemia virus (EIAV) called vMA-1c that rapidly and specifically killed infected equine fibroblasts (ED cells) but not other infectible cell lines was established. This strain was generated from an avirulent, noncytopathic strain of EIAV, MA-1. Studies with this new cytolytic strain of virus have permitted us to define viral parameters associated with EIAV-induced cell killing and begin to explore the mechanism. vMA-1c infection resulted in induction of rapid cell death, enhanced fusogenic activity, and increased rates of spread in equine fibroblasts compared...
Ghallab AM, Shahat AM, Fadl AM, Ayoub MM, Moawad AR.The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5-ml plastic straws. After cooli...
Durgam SS, Stewart AA, Pondenis HC, Yates AC, Evans RB, Stewart MC.To compare in vitro expansion of equine tendon- and bone marrow-derived cells with fibroblast growth factor-2 (FGF-2) supplementation and sequential matrix synthesis with pulverized tendon and insulin-like growth factor-I (IGF-I). Methods: Cells from 6 young adult horses. Methods: Progenitor cells were expanded in monolayers with FGF-2, followed by culture with autogenous acellular pulverized tendon and IGF-I for 7 days. Initial cell isolation and subsequent monolayer proliferation were assessed. In pulverized tendon cultures, cell viability and expression of collagen types I and III and carti...
Catalán J, Llavanera M, Bonilla-Correal S, Papas M, Gacem S, Rodríguez-Gil JE, Yeste M, Miró J.The aim of this study was to evaluate whether red-light stimulation increases the longevity and resilience of cryopreserved stallion sperm to withstand post-thaw incubation for 120 min. Sixteen frozen straws of 0.5 mL from eight stallions were used. Samples were cryopreserved, thawed through incubation at 38 °C for 30 s and divided into the control and samples exposed to red-light using a triple LED photo-activation system (wavelength: 620-630 nm). Three irradiation protocols consisting of different light-dark-light intervals (1-1-1, 2-2-2 and 3-3-3 min) were tested. Sperm quality param...
Alamaary MS, Haron AW, Ali M, Hiew MWH, Adamu L, Peter ID.Different types of extenders have a variety of components which show the tolerance effect on sperm protection during freezing procedures. In the present study, we have examined the impact of the extenders HF-20 and Tris, which were locally manufactured, and they are competing with commercial extenders INRA Freeze® (IMV Technologies, France) and EquiPlus Freeze® (Minitube, Germany) on the quality of horses frozen semen. Methods: A total of 15 ejaculates from three healthy stallions were collected and cryopreserved in the same environment. Each semen sample collected was divided into four equa...
Bucci D, Giaretta E, Spinaci M, Rizzato G, Isani G, Mislei B, Mari G, Tamanini C, Galeati G.Alkaline phosphatase (AP) has been studied in several situations to elucidate its role in reproductive biology of the male from different mammalian species; at present, its role in horse sperm physiology is not clear. The aim of the present work was to measure AP activity in seminal plasma and sperm extracts from freshly ejaculated as well as in frozen-thawed stallion spermatozoa and to verify whether relationship exists between AP activity and sperm quality parameters. Our data on 40 freshly ejaculated samples from 10 different stallions demonstrate that the main source of AP activity is semi...
Shivaram A, Bose S, Bandyopadhyay A.The primary objective of this research was to evaluate the extent of mechanical degradation on TiO2 nanotubes on Ti with and without nano-particulate silver coating using two different lengths of TiO2 nanotubes-300nm and ~1µm, which were fabricated on commercially pure Titanium (cp-Ti) rods using anodization method using two different electrolytic mediums-(1) deionized (DI) water with 1% HF, and (2) ethylene glycol with 1% HF, 0.5wt% NH4F and 10% DI water. Nanotubes fabricated rods were implanted into equine cadaver bone to evaluate mechanical damage at the surface. Silver was electrochemical...
Ball BA, Vo A.Osmotic stress attributed to differences in the relative permeability of cryoprotectants, such as glycerol and water, appears to be an important factor in cryodamage. The objective of this study was to characterize the osmotic tolerance of equine spermatozoa, and to evaluate the effects of addition and removal of cryoprotectants from equine spermatozoa on their motility, and membrane and acrosomal integrity, as well as their mitochondrial membrane potential. Equine spermatozoa had a limited osmotic tolerance to anisosmotic conditions. Although the addition of increasing concentrations of glyce...
Grady ST, Britton L, Hinrichs K, Nixon AJ, Watts AE.Mesenchymal stem cells (MSCs) improve the osteoarthritis condition, but the fate of MSCs after intra-articular injection is unclear. We used fluorescent nanoparticles (quantum dots [QDs]) to track equine MSCs (QD-labelled MSCs [QD-MSCs]) in vivo after intra-articular injection into normal and osteoarthritic joints. One week after injection of QD-MSCs, unlabelled MSCs, or vehicle, we determined the presence of QD-MSCs in synovium and articular cartilage histologically. In vitro, we evaluated the persistence of QDs in MSCs and whether QDs affected proliferation, immunophenotype, or differentiati...
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 ...
Benbarek H, Deby-Dupont G, Caudron I, Grülke S, Deby C, Lamy M, Serteyn D.In horses, the mechanisms of lipopolysaccharide (LPS) stimulation of isolated neutrophils to produce reactive oxygen species remain unknown. We re-investigated this problem by monitoring the luminol-enhanced chemiluminescence (CL) produced by LPS-stimulated equine neutrophils. The neutrophils were isolated from horse blood by discontinuous density gradient centrifugation (> or = 99% neutrophils; viability > or = 98%). Increasing concentrations of Escherichia coli (E. coli) LPS (from 0.01-10 microg ml(-1)) were used to activate the neutrophils. When LPS was used directly, without another ...
Duan W, Chen C, Haque M, Hayes D, Lopez MJ.Use of bioscaffolds to direct osteogenic differentiation of adult multipotent stromal cells (MSCs) without exogenous proteins is a contemporary approach to bone regeneration. Identification of in vivo osteogenic contributions of exogenous MSCs on bioscaffolds after long-term implantation is vital to understanding cell persistence and effect duration. This study was designed to quantify in vivo equine MSC osteogenesis on synthetic polymer scaffolds with distinct mineral combinations 9 weeks after implantation in a murine model. Cryopreserved, passage (P)1, equine bone marrow-derived MSCs (BMSC)...
Jörg A, Portmann P, Fellay G, Dreyer JL, Meyer J.An improved and short method is described for the isolation of intact eosinophilic leukocytes from horse blood with high yield (1--1.5 g/20 l). Viability and purity of the preparations were verified by light and electron microscopy and by the trypan blue exclusion test. Isolated eosinophils were 98--100% pure, intact and viable, and they could be shown to phagocytise immune-complexes.
Violini S, Gorni C, Pisani LF, Ramelli P, Caniatti M, Mariani P.Stem cells represent an important tool in veterinary therapeutic field such as tissue engineering. In the present study, equine amnion-derived mesenchymal stromal cells were investigated for applications in veterinary science as an alternative source to bone marrow mesenchymal stem cells and adipose stem cells. Amnion stromal cells isolation and characterization protocol is described; the in vitro cell growth rate was calculated by measuring viable cell number over 20 days. The expression of stem cell markers such as Oct-4, Nanog, Sox-2 and CD105 was assessed by retrotranscription quantitativ...
Hoffmann N, Oldenhof H, Morandini C, Rohn K, Sieme H.Cryopreserved stallion sperm displays a high degree of male-to-male variability with respect to cell viability after thawing. Animals that have semen with low viability after cryopreservation are classified as 'poor' freezers, and when post-thaw viability is high they are designated as 'good' freezers. Cryoprotective agents that are used for cryopreserving stallion sperm include glycerol, ethylene glycol, methyl formamide, and dimethylformamide, and are typically used in concentrations ranging from 1% to 4%. The aim of this study was to evaluate the osmotic stresses that stallion sperm is expo...
Mancini F, Nannarone S, Buratta S, Ferrara G, Stabile AM, Vuerich M, Santinelli I, Pistilli A, Chiaradia E.To assess the effects of xylazine and dexmedetomidine on equine chondrocytes, in vitro. Methods: Prospective, experimental study. Methods: Equine articular chondrocytes from five male horses. Methods: Chondrocytes were isolated from healthy equine articular cartilage of the metacarpo/metatarsophalangeal joints. Cell viability was assessed using the WST-8 assay by exposing chondrocytes to xylazine (0.5, 1, 2, 4, 8, 16.6, 25, 50 mg mL) or dexmedetomidine (0.001, 0.005, 0.01, 0.05, 0.175, 0.25 mg mL) for 15, 30 and 60 minutes. Based on the results of these tests, cells were treated with xyla...
Costa GMJ, Avelar GF, Lacerda SMSN, Figueiredo AFA, Tavares AO, Rezende-Neto JV, Martins FGP, França LR.The establishment of proper conditions for spermatogonial stem cells (SSCs) cryopreservation and storage represents an important biotechnological approach for the preservation of the genetic stock of valuable animals. This study demonstrates the effects of different cryopreservation protocols on the survival rates and phenotypic expression of SSCs in horses. The cells were enzymatically isolated from testes of eight adult horses. After enrichment and characterization of germ cells in the suspension, the feasibility of several cryopreservation protocols were evaluated. Three different cryomedia...
Byron CR, Benson BM, Stewart AA, Stewart MC.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...
Marlo TL, Giuliano EA, Sharma A, Mohan RR.To develop an ex vivo equine corneal organ culture model. Specifically, to assess the equine cornea's extracellular matrix and cellularity after 7 days using two different culture techniques: either (i) immersion system or (ii) air/liquid interface system, to determine the best ex vivo equine corneal model. Methods: Fourteen healthy equine corneas of various breeds. Methods: Equine corneas with 2 mm of perilimbal sclera were freshly harvested from 7 horses undergoing humane euthanasia. One corneal-scleral ring (CSR) from each horse was randomly placed in the (i) immersion condition organ cultu...
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...
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 ...
Zayed M, Adair S, Dhar M.Synovial fluid contains cytokines, growth factors and resident mesenchymal stem cells (MSCs). The present study aimed to (1) determine the effects of autologous and allogeneic synovial fluid on viability, proliferation and chondrogenesis of equine bone marrow MSCs (BMMSCs) and (2) compare the immunomodulatory properties of equine synovial fluid MSCs (SFMSCs) and BMMSCs after stimulation with interferon gamma (INF-γ). To meet the first aim of the study, the proliferation and viability of MSCs were evaluated by MTS and calcein AM staining assays. To induce chondrogenesis, MSCs were cultured in ...
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...
Masri M, Lombardero G, Velasquillo C, Martínez V, Neri R, Villegas H, Ibarra C.The goal of this study is to evaluate the efficiency of obtaining a large number of viable cells within a construct that will not be detached by high fluid flow during arthroscopic implantation. Methods: Arthroscopic osteochondral biopsy specimens were obtained from the medial femoral trochlea of 8 horses. Chondrocytes were isolated by collagenase digestion and expanded in M199 media until confluency. After 10 to 12 days, cultures were trypsinized and cells resuspended in culture media. Then, 5 x 10(6) cells x mL(-1) were seeded on a culture dish and the same amount in a flask. Once extracellu...
de Leon PM, Campos VF, Corcini CD, Santos EC, Rambo G, Lucia T, Deschamps JC, Collares T.The objective was to evaluate the effect of three cryopreservation methods on the in vitro maturation (IVM) and membrane integrity (MIn) of immature equine oocytes. An open pulled straw (OPS) method, a novel solid surface vitrification (SSV) process, and the addition of a synthetic ice blocker were evaluated. Compared with the control group (N=269), the OPS (N=159) and the SSV (N=202) cryopreservation methods decreased both IVM (50.9 vs. 13.3 and 9.4%, respectively; P<0.001) and MIn (76.6 vs. 31.1 and 33.7%; P<0.001) of immature equine oocytes. However, inclusion of 0.1% ice blocker in t...
