In vitro research involving horses refers to the study of equine cells, tissues, or biological molecules outside their normal biological context, typically in controlled laboratory environments. This research approach allows scientists to investigate cellular processes, molecular interactions, and the effects of various treatments without the ethical and logistical complexities of in vivo studies. In vitro studies contribute to understanding equine physiology, pathology, and pharmacology by providing insights into cellular responses to pathogens, drugs, and other stimuli. This page compiles peer-reviewed research studies and scholarly articles that explore various in vitro methodologies and their applications in equine science, including cell culture techniques, molecular assays, and drug efficacy testing.
SASAKI MS, SASAKI M.In two tissue culture lines, HS and HF, which originated from foetal horse tissues, the changes of chromosomes were studied in the course of serial in vitro transfers. It was found that the two cell lines under consideration were characterized by specific changes in chromosome pattern different from each other. HS underwent transformation from diploidy to hyperdiploidy. HF may have been in a process of transformation from diploidy to tetraploidy. Evidence derived from this study indicates that several courses for chromosome transformation exist, and that generalizations predicting a single pat...
REBERS PA, HURWITZ E, HEIDELBERGER M.Rebers, Paul A. (Rutgers University, New Brunswick, N. J.), Esther Hurwitz, and Michael Heidelberger. Immunochemistry of pneumococcal types II, V, and VI. II. Inhibition tests in the type VI precipitating system. J. Bacteriol. 82:920-926. 1961.-As in other immune systems involving polysaccharides, rabbit antibodies but not those engendered in the horse were found sensitive to degradation of type VI pneumococcal (Pn) polysaccharide (SVI), and were readily inhibited by fragments of SVI. Large amounts, 30 to 111 mumoles, of most sugars gave up to 15% inhibition, while sugar and polyol phosphates ...
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Frontiers in geneticsJuly 18, 2025
Volume 16 1630614 doi: 10.3389/fgene.2025.1630614
Gong W, Ding W, Bou T, Shi L, Lin Y, Shi X, Li Z, Wu H, Dugarjaviin M, Bai D.Ferulic acid (FA), a natural antioxidant, has attracted considerable attention for its regulatory potential in skeletal muscle development, energy metabolism, and muscle fiber type transformation. Unassigned: This study established a research system based on Mongolian horse skeletal muscle satellite cells to elucidate the molecular basis by which ferulic acid regulates muscle fiber type transformation through a non-coding RNA interaction network. Unassigned: A total of 18 differentially expressed miRNAs (DEMIRs) and 128 differentially expressed lncRNAs (DELs) were identified through transcript...
Palomino Lago E, Roberts SJ, Orriss IR, Lumsden ES, Guest DJ.Bone is a dynamic tissue that is maintained through the co-ordination of bone resorption and bone formation. An imbalance of these processes can lead to bone disease. In vitro studies of osteoblasts can help to understand bone formation, but primary cells have a limited lifespan in culture. Herein, we report the successful generation of equine immortalized osteoblasts through the stable overexpression of human telomerase reverse transcriptase (hTERT) and Simian virus 40 (SV40) large T-antigen in osteoblasts isolated from trabecular bone taken from the third metacarpal of a two-year-old Thoroug...
Young KAS, Schnabel LV, Gilger BC.Equine ocular disease is common and often challenging to treat using traditional methods. This has led to the development of new therapies. Like human medicine, veterinary medicine is adopting cellular and gene therapy as innovative approaches. Equine ocular disease is a particularly promising area for these techniques. Notably, immune-mediated diseases (such as immune-mediated keratitis and equine recurrent uveitis), ulcerative keratitis, and infectious ocular diseases are of interest. Several ocular gene therapy products are approved for use in humans, and more are currently being researched...
Cortez JV, Cervi D, Ruiz AJ, Grupen CG.Nicotinic acid (NA) treatment during in vitro maturation (IVM) has been shown to elevate nicotinamide adenine dinucleotide (NAD) levels and improve oocyte developmental competence. Suboptimal equine oocyte IVM systems currently limit the efficiency of viable embryo in vitro production. This study evaluated NA supplementation during IVM for cloned equine embryo production, using oocytes from abattoir-sourced ovaries and live mares via ovum pick-up (OPU). Abattoir-derived oocytes (n = 694) were treated without or with 50 or 200 μM NA during the 18 h holding period (Pre-IVM). Next, OPU-deriv...
Castillo JDCR, Forte L, Elghandour MMMY, Maggiolino A, De Palo P, Ponce-Covarrubias JL, Salem AZM.Parasitic infections remain a major constraint to equine health and performance, and the worldwide rise of anthelmintic resistance is forcing a shift toward integrated parasite management and new complementary tools. Caesalpinia coriaria (Divi-divi) pods are exceptionally rich in tannins and have been proposed as a potential source of antiparasitic phytochemicals. This review summarizes the current evidence on C. coriaria, focusing on its relevance to equine gastrointestinal helminths. Most available data come from in vitro screening against ruminant nematode models (mainly Haemonchus contortu...
Díaz-Alvarado T, Alcala-Canto Y, Elghandour MMMY, Salem MZM, Mariezcurrena-Berasain MD, Salem AZM.Gastrointestinal endoparasitic diseases, particularly those caused by some strongyles, represent one of the main health challenges in equine production. The prolonged use of synthetic anthelmintics has led to the development of resistance, which has prompted the search for more sustainable therapeutic alternatives. However, plant extracts rich in secondary metabolites are emerging as a promising option. Objective: The objective of this study was to evaluate the in vitro anthelmintic activity of aqueous extracts and bioactive compounds (total phenols and saponins) from Azadirachta indica and Cn...
Tsopp E, Kilk K, Gambini A, Tvrdà E, Viljaste-Seera A, Tuppits U, Jaakma Ü.Equine in vitro embryo production (IVP) via ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) has gained prominence in sport-horse breeding due to high success rates, efficient semen utilization, and year-round embryo production, including from mares with fertility challenges or postmortem. However, assessing viability of early-stage ICSI-produced equine embryos remains challenging due to subjective morphological evaluations and limited visibility of developmental features. Non-invasive metabolomic analysis of culture media offers a promising alternative to enhance embryo selectio...
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...
Medica AJ, Gibb Z, Ortiz-Rodriguez JM, Swegen A.Heavy use of antioxidants in equine in vitro fertilisation (IVF) media may suppress the reactive oxygen species (ROS) signals stallion sperm need for capacitation, which depends on high mitochondrial activity and a well-maintained endogenous redox homeostasis. A species-specific approach balancing oxidative protection with controlled ROS signalling could improve fertilisation efficiency and reduce prolonged incubation times.
Nunes MM, Gurgel JRC, Munhoz ML, de Giuli V, Carneiro GF, Miragaya MH, Gallelli MF, Guimaraes JD.The aim of this study was to assess the effect of lyophilized freezing extenders, which can be stored at room temperature, on stallion post-thaw sperm total motility (TM). Ejaculates of 28 stallions were frozen with four different extenders: two commercial freezing extenders offered worldwide and two novel lyophilized extenders (STAR and MX3), and two different cryopreservation protocols (CP1 with an equilibration period of 20 min. and CP2 with an equilibration period of 60 min.). The TM was assessed after thaw. Mean TM did not show significant differences between cryopreservation protocols wi...
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...
Ramírez-Agámez L, Castaneda C, Hernández-Avilés C, Grahn RA, Raudsepp T, Love CC.Two methods for preimplantation genetic testing (PGT) have been described for equine embryos: trophoblast cell biopsy (TCB) or blastocoele fluid aspiration (BFA). While TCB is widely applied for both in vivo- and in vitro-produced embryos, BFA has been mostly utilized for in vivo-produced embryos. Alternative methods for PGT, including analysis of cell-free DNA (CFD) in the medium where in vitro-produced embryos are cultured, have been reported in humans but not for equine embryos. In Experiment 1, in vivo- (n = 10) and in vitro-produced (n = 13) equine embryos were subjected to BFA, cultu...
Mazzuchini MP, Lisboa FP, Segabinazzi LG, Canisso IF.Endometritis is the leading cause of mare subfertility. Most mares respond to standard therapy, but alternative therapies have been developed for mares failing to respond. This study aimed to investigate a commercially available, yet unassessed, product labeled as a uterine sanitizer to determine the in vitro antimicrobial activity against microorganisms associated with endometritis and its in vitro stability to dilute antibiotics. In experiment 1, the microdilution broth technique and antimicrobial effects were assessed against Escherichia sp, Staphylococcus sp., Klebsiella sp., Pseudomonas s...
Caldevilla M, Ferrante A, Neild DM.The addition of antioxidants to cryopreservation media reportedly improves sperm post-thaw quality and reproductive performance after artificial insemination. Therefore, the objectives of this study were to evaluate if the addition of L-carnitine and pyruvate to freezing media, or their addition to samples after thawing, improves the post-thaw quality of equine spermatozoa. Thus, in Experiment 1, stallion semen samples were cryopreserved in: (1) EDTA-glucose-based extender with 20% egg yolk and 5% dimethylformamide (EDTA control); (2) skim milk-based extender with 20% egg yolk and 5% dimethylf...
Arnold LC, Stout TAE, Beitsma MM, Deelen CC, Rizzo M, Leemans B, Galli C, Colleoni S, Lazzari G, Barandalla M, Claes ANJ.It is not known whether the likelihood of pregnancy after transfer of cryopreserved equine in vitro produced (IVP) blastocysts is related to post-thaw morphological characteristics, or could be impacted by other factors such as embryo biopsy prior to cryopreservation. This study examined associations between post-thaw morphological appearance of equine IVP embryos, and biopsy prior to slow-freezing, on the likelihood of pregnancy. Biopsy of IVP blastocysts was performed by harvesting cells herniated through the intra-cytoplasmic sperm injection (ICSI)-derived hole in the zona pellucida (ZP). M...
Pezzanite LM, Nelson BB, Downey AC, Gadomski B, McGilvray K, Baer K, Kappel SM, Nout-Lomas Y, Seim HB, Easley JT.To determine the biomechanical properties of pedicle screw and rod (PSR) constructs alone and with an interbody fusion device (PSRIFD) for equine ventral cervical vertebral stabilization. Methods: Cadaveric ex vivo biomechanical analysis. Methods: A total of 14 (n = 14) adult equine cervical vertebral columns. Methods: Cervical vertebral columns were stabilized by PSR alone (n = 6) or PSRIFD (n = 5). Three columns were left unaltered as controls. Non-destructive biomechanical testing showed the kinematic range of motion (ROM), compliance, and neutral zone of each spinal unit in the...
Dubeibe-Marin DF, Borraez DF, Gomez-López DL, Velasco-Acosta DA.This study evaluated the effect of the presence and location of the corpus luteum (CL) on quantity and competence of equine cumulus-oocyte complexes (COCs). Pairs of ovaries were collected at the slaughterhouse and classified into three groups: ovaries with CL (CL+); ovaries with CL in the contralateral ovary (CL-) and ovaries without a CL (NCL). Were evaluated COC quantity, morphology, nuclear maturation rate, and intrafollicular progesterone concentration and gene expression (BAX, BMP-15, ODC1, GDF9, AURKA) in both cumulus cells and oocytes after in vitro maturation. Results showed a lower r...
Hoerdemann M, Sahoo DK, Allbaugh RA, Kubai MA.To assess if an affordable, commercially available 275 nm ultraviolet C (UV-C) device can inhibit fungal pathogens associated with equine keratomycosis and to establish the optimal exposure time/dose required in vitro. Methods: Fungal inhibition zone surface areas of isolates from two fungal genera (Aspergillus and Fusarium spp.) were compared in triplicates after UV-C exposure at distances of 10, 15, and 20 mm with single or repeat doses (4 h apart) of 5, 10, 15, or 30 s duration after 24, 48, and 72 h of incubation. Inhibition zones were microscopically assessed, and regrowth chec...
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...
Ramírez-Agámez L, Hernández-Avilés C, Samper JC, Love CC.Intracytoplasmic sperm injection (ICSI) using frozen/thawed (F/T) stallion sperm is a common procedure in the equine breeding industry. Historically, sex-sorted (SS) F/T stallion sperm has yielded lower cleavage (<30 %) and blastocyst rates (<5 %) after ICSI when compared to non-sorted (NS) F/T sperm. Recently, a new technology for sperm sex-sorting (Genesis III) has been validated by a commercial company. In Experiment 1, the post-thaw quality between NS-F/T and SS-F/T stallion sperm produced with this technology was compared. The post-thaw sperm motility was higher in NS-F/T (41 %) than i...
Silva GLS, Curcio UA, Boufleur JA, Borges DGL, Nakatani MTM, Freitas MG, Borges FA, Faria FJC, Silva RHP, Franco GL.Anthelmintic resistance has led to the use of organic extracts as alternative methods of parasite control. Objective: The study aimed to assess the effects of Acacia mearnsii extract (tannin) on the control of cyathostomins in horses, both in vitro and in vivo. Methods: Thirty Pantaneiro horses naturally infected with cyathostomins were sourced from two distinct farms, designated as Farms A and B. At the start of the study, all third-stage larvae were cyathostomins, and an egg hatchability test (EHT) was performed using fecal samples from horses on both farms. Two randomized 28-day field studi...
Hedia M, Leroy JLMR, Loomans S, Benedetti C, Angel-Velez D, Chiers K, Govaere J, Van Soom A, Smits K.Lipopolysaccharide (LPS) in follicular fluid impairs steroid production and oocyte developmental competence in cows and mice. This study assessed LPS concentrations in equine follicular fluid and their association with steroid and some cytokine levels. Additionally, we evaluated whether LPS exposure during in vitro maturation (IVM) affects equine oocyte developmental competence. In experiment 1, follicular fluid from large follicles (>30 mm in diameter) was collected from 16 slaughterhouse mares, and concentrations of LPS, estradiol, progesterone, TNF-α, and IL-6 were measured. In experiment...
Cloteau C, Delcourt V, Loup B, Chabot B, Pescher M, Susdorf E, Kaabia Z, Garcia P, Popot MA, Le Bizec B, Dervilly G, Bailly-Chouriberry L.Biomarker identification by mass spectrometry represents a key step in the workflow of nontargeted metabolomic studies. Given the complexity of the data, this step, which must be carried out by a trained specialist, is time-consuming, and the biomarkers discovered are not always identified. While this stage is not an obstacle to the development of new screening and classification tools, it is nonetheless crucial to a better understanding of the results obtained. For this reason, the aim of this study was to perform structural elucidation of candidate biomarkers, which had previously been displ...
Zedler S, Jukic C, van Eps A, Stefanovski D, Genton M, Rossignol F.To compare the biomechanical properties of a three-dimensional (3D) printed titanium plate and spacer (3DM) and a 4.5 mm broad locking compression plate (LCP) for intervertebral fusion of C4-C5 in the horse. Methods: Ex vivo experimental study. Methods: Twenty-four equine cadaveric cervical spines. Methods: Twenty-four cadaver equine cervical spines were randomly assigned to ventral intervertebral fusion with either 3DM (n = 12) or LCP (n = 12) at C4-C5. Four-point bending tests were performed in flexion (3DM n = 6, LCP n = 6) and extension (3DM n = 6, LCP n = 6) in a sin...
Urayama S, Muko R, Muranaka M, Mita H, Ohta M, Matsuda H, Tanaka A.Systemic inflammatory response syndrome (SIRS) induced by endotoxemia is usually secondary to colitis and is a major cause of high morbidity and mortality in horses. Non-steroidal anti-inflammatory drugs (NSAIDs), such as flunixin meglumine (FM) and meloxicam (MX), are used to improve clinical outcomes in SIRS/endotoxemia. These NSAIDs suppress tumor necrosis factor-alpha (TNF-α) levels; however, the underlying mechanisms remain unclear. The aim of this study was to investigate the inhibitory effects of FM and MX on TNF-α in lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear ce...
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...
Broothaers K, Angel-Velez D, Molto FLG, Hedia M, Coster T, Govaere J, Soom AV, Menten B, Smits K.In 2022, a repeatable protocol for in vitro fertilization (IVF) using fresh semen was established in horses. This facilitated successful capacitation of equine semen allowing to explore novel applications. Objective: We aimed to extend this technique to IVF with frozen-thawed semen and intracytoplasmic sperm injection (ICSI), and determine the outcome parameters such as blastocyst production and euploidy rates. Methods: A total of 221 oocytes were subjected to either IVF with frozen-thawed semen, ICSI with frozen-thawed semen incubated under capacitating conditions (ICSI cap) or control ICSI w...
Salinas-Varas C, Espinosa G, Muñoz-Caro T, Conejeros I, Gärtner U, Fey K, Arnhold S, Taubert A, Hermosilla C.Neutrophil extracellular trap (NET) are thin and long web-like structures composed of DNA and antimicrobial proteins released by activated polymorphonuclear neutrophils (PMN) as part of the innate immune response. Adipose-derived stem cells (ADSCs) represent an accessible, abundant and minimal invasive source of mesenchymal stem cells (MSCs), with high regenerative potential, immunomodulatory and anti-inflammatory properties. Although recognized immunomodulatory properties of ADSCs, their interaction with PMN and their role on NET formation remains poorly characterized. The present study aimed...
Broothaers K, Jouneau A, Angel-Velez D, De Coster T, Festuccia N, Archilla C, Calderari S, Jouneau L, Van den Branden E, Peere S, Polfliet E....In vitro production of equine embryos has been performed using intracytoplasmic sperm injection (ICSI) for the last two decades. Since 2022, a repeatable protocol for conventional in vitro fertilization (IVF) provides a successful alternative. However, little is known about the influence of the fertilization method on embryo quality and the transcriptomic profile. In this study, we aimed to examine differentially expressed genes (DEGs) between ICSI and IVF embryos in the horse. Therefore, ten equine sibling blastocysts, produced in vitro by either ICSI or IVF from three different mares, were s...
