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.
Ferrante A, Thong YH.Results presented show that highly purified populations of mononuclear (MN) and polymorphonuclear (PMN) leucocytes can be obtained from horse blood by a procedure similar to that previously described for the separation of these leucocytes from human blood. This involved centrifugation of horse blood on a Ficoll-Hypaque medium with a density of 1.095 g/ml. The procedure required approximately 1 h for completion and resulted in the simultaneous preparation of MN (greater than 98% purity) and PMN (greater than 96% purity) leucocytes. Cell viability exceeded 95% and cells retained immunological fu...
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...
Kenney RM, Condon W, Ganjam VK, Channing C.The histological features and hormonal content of follicular fluid of antral follicles during oestrus were correlated. As a result it was possible to characterize several categories of viable and atretic follicles. A seemingly important stage in maturation appeared to be at 3 cm in diameter since follicular oestrogens and androgens underwent a 3-fold increase in concentration at that size. Evidence was obtained to suggest that oestrogens are anti-atretogenic. However, a drop in oestrogens was not the cause of atresia since degeneration commenced when levels were high. Contrary to the concept t...
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.
Scott EA, Thrall DE, Sandler GA.Vascular supply of the equine forelimb was partially disrupted by ligation of the medial palmar and medial palmar digital arteries. Vascular architecture was evaluated before and after ligation by angiography. It was apparent from the present study that existing collaterals or newly formed collaterals, or both, were sufficient to maintain limb viability.
Targowski SP.The present study describes a two step technique for the separation of mononuclear leukocytes or mononuclear and polymorphonuclear leukocytes from whole equine blood. First, the leukocyte rich plasma was obtained by sedimentation of erythrocytes in the undiluted blood. Subsequently, separation of the different populations of white blood cells was performed by centrifugation with different gradients overlaid with the leukocyte rich plasma. The optimal separation of the mononuclear cells was obtained by the centrifugation of the leukocyte rich plasma overlaying the gradient containing 24 parts o...
Folin M, Gennari G, Jori G.The irradiation of horse and sperm-whale Fe” or Fez’ myoglobins with visible light
showed that axial ligands that render the heme diamagnetic (e.g. 02, CO or CN-) endow the
hemoproteins with a marked photosensitivity. In contrast, high-spin myoglobins are unaffected by
visible light. These findings appear to be of general validity for all hemo-proteins and are in agreement
with the involvment of the triplet state of the heme as the reactive intermediate. In all cases, the overall
photoprocess occurs within a very narrow spatial range, leading to specific modification of these
photoox...
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...
Boone CW, Harell GS, Bond HE.Large-scale separation of mixtures of mammalian cells was obtained with the A-1X zonal centrifuge rotor and density gradients consisting of Ficoll dissolved in modified Eagle's MEM suspension-culture medium. The cells remained viable as tested by plating efficiency or by motility observed with time-lapse photography. Rabbit thymocyte and HeLa cell mixtures were separated with 99 and 89 per cent purity, respectively. Mixtures of thymocytes and suspension-cultured, human acute leukemia cells (Roswell Park strain LKID) were separated with 93 and 91% purity, respectively. HeLa cells were isolated ...
Hernández-Avilés C.Sperm quality analysis using computer-assisted sperm analysis (CASA) systems and fluorescence-based techniques has become common in the animal reproduction industry, particularly for large animals (i.e., bovine, porcine, equine). In this chapter, the methods commonly utilized in the author's laboratory to examine sperm motion characteristics via CASA and plasma membrane intactness by flow cytometry will be described. These include methods to properly dilute fresh (stallions, bulls, boars), cool-stored (stallions, boars), or frozen/thawed (stallions, bulls, boars) sperm for assessment of sperm ...
Semik-Gurgul E, Ocłoń E, Zubel-Łojek J, Pędziwiatr R, Pawlina-Tyszko K.Sarcoids are locally invasive skin tumors in equids, associated with bovine papillomavirus. Objective: Address potential applications of cannabidiol (CBD) in veterinary medicine. We evaluated the response of equine sarcoid cells to CBD in vitro, focusing on viability, invasiveness, and matrix remodeling. Methods: Three primary sarcoid cell lines. Methods: Cells were treated with CBD (20, 6.75, 2.25, 0.75 μM) and incubated for 6, 24, 48, 72 hours. Cell viability, cytotoxicity, and apoptosis were assessed using the ApoTox-Glo Assay. Based on these results, further analyses were performed for ...
Normélia P Morais A, Gastal GDA, Aguiar FLN, Hyde KA, Gastal MO, Lima LF, Ñaupas LVS, Flávia B Silva A, Becerra VAB, Gheno L, Curcio BR....Understanding how to effectively isolate a large number of healthy follicles is essential for increasing success rates in ARTs, such as in vitro culture and cryopreservation. Late secondary (preantral) and early tertiary (antral) follicles isolated from the intermediary portion of equine ovarian tissue sliced at two different thicknesses (0.25 mm vs. 0.50 mm) were characterized according to regional distribution (dorsal, medial, and ventral), morphofunctional features, and epigenetic profiles. After isolation via microdissection, follicles were evaluated for membrane integrity (trypan blue), v...
Kushida C, Tamura N, Kasashima Y, Sato K, Arai K.In equine regenerative medicine using bone marrow-derived mesenchymal stem/stromal cells (BM-MSC), the importance of the quality management of BM-MSC has been widely recognized. However, there is little information concerning the relationship between cellular senescence and the stemness in equine BM-MSC. In this study, we showed that stemness markers (NANOG, OCT4, SOX2 and telomerase reverse transcriptase) and colony forming unit-fibroblast apparently decreased accompanied with incidence of senescence-associated β-galactosidase-positive cells by repeated passage. Additionally, we suggested th...
Spacek SG, Hernández-Avilés C, Ramírez-Agámez L, Pearson M, Love CC.Cryopreservation of stallion epididymal sperm has become a common clinical procedure after routine castration, euthanasia, or acute death. Unique features of epididymal sperm compared to ejaculated sperm include the requirement to remove sperm from the cauda epididymis, lack of exposure to seminal plasma, and potential recovery of large sperm numbers. In this study, the effect of the flushing technique (Extender (INRA) vs. AIR), freezing extender (LE, CMLE, MFR5, CFR5, or BOTU), and the concentration at which sperm are cryopreserved (200, 400, or 800 × 10 sperm/mL) on post-thaw epididymal s...
Alves LSS, Hasuda AL, Giordano LGP, Frederico I, Dos Santos IH, Pereira PFV, Lisbôa JAN, Flaiban KKMDC.This study investigated isolation of equine neutrophils and the assessment of their antioxidant function using the nitroblue tetrazolium (NBT) reduction test, an indicator of reactive oxygen species (ROS) activation. The objective was to test the viability of neutrophils isolated from equine whole blood and their ability to undergo respiratory burst, comparing the results with whole blood. The hypothesis tested was that isolation of equine neutrophils allows for effective functional evaluation, even after isolation process, with no significant differences between whole blood and isolated cells...
Zamboulis DE, Thorpe CT.The high stresses and strains experienced by the superficial digital flexor tendon (SDFT) make it prone to age-related injury. The SDFT exhibits cellular and structural specializations, localized to the interfascicular matrix (IFM), enabling it to resist its extreme mechanical environment. With aging, these specializations are lost, impacting the ability of the tendon to withstand repeated loading. Within the IFM, there is loss of mechanical properties and organization accompanying cell senescence and a decreased resolution of inflammation. Developing therapeutics to reverse these changes may,...
Sawicki S, Bugno-Poniewierska M, Żurowski J, Szmatoła T, Semik-Gurgul E, Bochenek M, Karnas E, Gurgul A. Mesenchymal stem cells (MSCs) are a promising tool in regenerative medicine due to their ability to secrete paracrine factors that modulate tissue repair. Extracellular vesicles (EVs) released by MSCs contain bioactive molecules (e.g., mRNAs, miRNAs, proteins) and play a key role in intercellular communication. This study compared the transcriptomic profiles (mRNA and miRNA) of equine MSCs derived from adipose tissue (AT-MSCs), bone marrow (BM-MSCs), and ovarian fibroblasts (as a differentiated control). Additionally, miRNAs present in EVs secreted by these cells were characterized using nex...
Bourebaba N, Domagała J, Bourebaba L.Equine metabolic syndrome (EMS) is associated with chronic low-grade inflammation, disruptions in mitochondrial dynamics and function, and an increased risk of developing laminitis. Recent research has highlighted that reduced levels of sex hormone-binding globulin (SHBG) in the bloodstream are linked to higher susceptibility to obesity, insulin resistance, and diabetes, potentially contributing to broader metabolic imbalances. This study aimed to evaluate whether exogenously administered SHBG could protect adipose-derived stem cells from horses affected by EMS (EqASC) against mitochondrial dy...
Bungărdean D, Pall E, Daradics Z, Popescu M, Tripon MA, Lupșan AF, Crecan CM, Morar IA, Nicolescu A, Bora FD, Marcus I.Musculoskeletal disorders are a major cause of lameness in horses, often necessitating innovative regenerative strategies to restore joint function and improve quality of life. This study investigated the effects of platelet-rich plasma (PRP), ozonized PRP, hyaluronic acid, paracetamol, and polyacrylamide hydrogel (NOLTREX) on the behavior of mesenchymal stem cells (MSCs) derived from equine synovial fluid. Synovial fluid samples were collected under strict cytological criteria to ensure viability, followed by in vitro expansion and phenotypic characterization of MSCs. Cultures were supplement...
Sabzpoosh M, Hoveizi E, Gooraninejad S.This study aimed to extract and isolate endometrial stromal cells from Arabian mares and investigate their growth and differentiation potential. Endometrial biopsies were obtained from three healthy 6-year-old Arabian mares using a standardized, minimally invasive protocol. The isolated cells were characterized using flow cytometry and differentiation analysis. Flow cytometry revealed mesenchymal markers CD90 (95.2%) and CD105 (97.4%) and hematopoietic markers CD34 (1.17%) and CD45 (0.339%). The cells exhibited differentiation potential into adipocytes, osteoblasts, and chondrocytes. The findi...
Wójtowicz A, Sadowska A, Myszczyński K, Molcan T, Kaczmarek MM, Szóstek-Mioduchowska A.Fibrosis remains incompletely understood, particularly in terms of how immune mediators shape stromal programs. We used a spontaneous large‑animal model-endometrosis (equine endometrial fibrosis) to define how interleukin‑4 (IL‑4) reprograms fibroblasts from healthy and fibrotic endometrium. Primary fibroblasts were exposed to IL‑4 (10 ng/mL) for 48 or 96 h. At 48 h, bulk transcriptomes revealed 1307 differentially expressed genes (DEGs; 648 up, 659 down) and 1271 DEGs (645 up, 626 down) in fibroblasts derived from endometria without or with endometrosis, respectively. Enrichment an...
Zanacchi A, Fruscione F, Grasselli S, Razzuoli E, Pastorino L, Di Lisa D.Developing advanced preclinical in vitro cancer models is essential for understanding tumor biology, improving drug discovery, and enhancing the clinical translation of new therapies. Papillomaviruses (PVs) cause diverse lesions in humans and animals; notably, bovine papillomavirus (BPV) is the main cause of equine sarcoid, the most common skin tumor in horses. Its pathogenesis involves viral infection, fibroblast transformation, and extracellular matrix (ECM) remodeling. These processes highlight the limitations of conventional two-dimensional (2D) culture systems, which fail to replicate the...
Parker JL, Berghaus LJ, Gordon J, Mathews L, Akinde MA, Hall DB, Peroni JF.Mesenchymal stromal cells (MSCs) support wound healing through immunomodulation and by promoting tissue organization. Cell tracking studies demonstrate that MSCs must 'home' to injuries in order to participate in reparative processes. It has been reported that in vivo cell tracking labels, including superparamagnetic iron oxide particles (SPIOs), can alter MSC homing. Homing is thought to occur in coordinated steps including tethering, activation, arrest, diapedesis, and migration, guided by chemotactic stimuli. Equine distal limb injuries, which are commonly treated with MSCs, possess a disti...
Del Prete C, Gaspari G, Kosior MA, Merlo B, Iacono E, Longobardi C, Martino NA, Dell'Aquila ME, Damiano S, Cocchia N, Gasparrini B, Lange-Consiglio A.Over the years, regenerative therapies have emerged as promising alternatives for persistent breeding-induced endometritis. In vitro studies testing the effects of these therapies on equine endometrial cells are still scarce. This study aimed to evaluate in vitro the effect of Wharton's jelly (WJ) mesenchymal stromal/stem cell (MSCs)-derived conditioned medium (WJ-CM) and platelet-rich plasma (PRP) on equine endometrial cells, with or without lipopolysaccharide (LPS)-induced inflammation. The WJ-CM was obtained after 24 h of starvation in Ringer's lactate of WJ-MSCs and PRP was prepared using...
DI Stefano DA, Coccoluto L, Panina-Bordignon P, Brambilla E, Ruffini F, Murtaj V, Orlando F, Colombo M, Frigerio C, DI Bona A, Recupero D, Morroni M....Bone grafting is a surgical procedure that involves the use of bone tissue or bone substitutes to repair damaged bone. In dentistry and maxillofacial surgery, bone graft substitutes from various sources are commonly used. Given their critical role in clinical outcomes, it is essential to thoroughly investigate the biological and mechanical properties of these materials. Methods: In this in vitro study, we evaluated the biological properties of two equine-derived bone graft substitutes in comparison to β-tricalcium phosphate. The materials included one equine-derived graft containing hydrolyze...
Fernandes CM, Prestes AS, Ianiski LB, Maciel AF, Noro BG, da Silva FD, Vizzotto BS, Botton SA, Schumacher RF, Pereira DIB, Barbosa NV.Pythiosis, caused by Pythium insidiosum, is an infectious and non-transmissible disease affecting horses, dogs, and humans, with no effective drug treatment available. Triazoles are compounds of interest for their potential pharmacological properties against fungi and bacteria. In this study, we synthesized three new triazole compounds (C1, C2, and C3) to assess their in vitro activities against P. insidiosum and their safety on human leukocytes. Susceptibility testing was performed against P. insidiosum isolates (n = 15) to determine the minimum inhibitory concentration (MIC) and minimum ...
