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Topic:In Vitro Research
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.
Isolation of major bacterial species associated with equine skin wounds and in-vitro antibacterial activities of selected medicinal plants. Wounds on the skin are a common health issue affecting working equines. This study aimed to evaluate the in-vitro antibacterial properties of crude methanolic extracts from selected medicinal plants against pathogens isolated from equine skin wounds in Merti district. Agar well and disc diffusion tests were used to determine the mean zone of inhibition, while broth dilution methods were used to determine the minimum inhibitory concentrations and minimum bactericidal concentrations (MBCs), further confirming the potent antibacterial effects of the selected medicinal plant extracts. One way anal...
Extracellular Vesicles Derived From Endometrial Stem Cells Preconditioned With PGE2-Reverse Myofibroblast Phenotype in Mare Endometrial Cells: A Novel Anti-Fibrotic Approach. Endometrial fibrosis in mares compromises fertility through aberrant extracellular matrix deposition and sustained myofibroblast activation. Conventional interventions fail to reverse these pathological alterations, necessitating innovative, mechanism-focused therapies. In this study, we pioneered the use of prostaglandin E2 (PGE2) preconditioning of equine endometrial-derived mesenchymal stem cells (ET-eMSCs) and their extracellular vesicles (EVs) to target fibrotic processes directly. ET-eMSCs were isolated from mare endometrial biopsies pretreated with PGE2 to enhance their anti-fibrotic se...
Isolation and characterization of mesenchymal stromal cells derived from equine ovarian follicular aspirates. The use of mesenchymal stromal cells (MSCs) in equine reproduction is increasing its interest in the treatment of specific pathologies. MSCs have been isolated from follicular aspirates obtained during transvaginal oocyte aspiration in women, offering a novel source for autologous therapies in reproductive treatments. However, this approach has not been tested in mares despite the common use of transvaginal oocyte aspiration for oocyte collection to produce equine embryos in vitro. Our study aimed to investigate the feasibility of isolating MSCs from equine ovarian follicular aspirates obtaine...
Comparative Study of Reprogramming Efficiency and Regulatory Mechanisms of Placental- and Fibroblast-Derived Induced Pluripotent Stem Cells (iPSCs) in Mules. As an interspecies hybrid inheriting genetic material from horse and donkey lineages, mules provide a unique model for studying allele-specific regulatory dynamics. Here, we isolated adult fibroblasts (AFs) and placental fibroblasts (PFs) from mule tissues and reprogrammed them into induced pluripotent stem cells (iPSCs). Intriguingly, placental fibroblast-derived iPSCs (mpiPSCs) exhibited reduced reprogramming efficiency compared to adult fibroblast-derived iPSCs (maiPSCs). Through allele-specific expression (ASE) analysis, we systematically dissected transcriptional biases in parental cell t...
Studies on in vitro production of equine embryos by Intracytoplasmic Sperm Injection (ICSI) using non-sorted, or sex-sorted, frozen/thawed stallion sperm: Effects on post-thaw sperm quality, cleavage and blastocyst rates, and characterization of cellular events during the first 24 hours post-fertilization via confocal microscopy. 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 %) ...
Abnormal cleavage patterns in equine in vitro-produced embryos lead to higher early pregnancy loss. Despite significant advances, in vitro production (IVP) of equine embryos continues to lack standardised embryo classification criteria and is associated with increased rates of early pregnancy loss compared with in vivo-derived blastocysts. Objective: To evaluate morphokinetic characteristics of the first mitotic division and early embryonic development in IVP blastocysts and their association with embryo development, as well as pregnancy rate and early pregnancy loss following embryo transfer. Methods: Retrospective analysis of archived material and clinical records. Methods: We retrospectiv...
Phenylbutazone treatment of mares does not reduce embryo production via intracytoplasmic sperm injection. Production of embryos via intracytoplasmic sperm injection (ICSI) is a common clinical procedure in equine practice. A recent study indicates that treatment of mares with phenylbutazone paste significantly lowers blastocyst production after ICSI of recovered oocytes. The current study was conducted to determine the effect of different methods of phenylbutazone administration on in vitro embryo production of recovered oocytes in mares. Unassigned: In study 1 (July 29 through September 18, 2024), 14 mares received either oral liquid phenylbutazone once daily for 10 days or no treatment. On the l...
Shifting Paradigms: Imidocarb dipropionate as an alternative chemotherapeutic strategy for Trypanosoma evansi infection in animals. We report on the in vitro and in vivo success of Imidocarb dipropionate (IDP), an aromatic diamidine compound, as an effective drug against Surra-causing organism, Trypanosoma evansi. IDP is a clinically acclaimed drug for the treatment of babesiosis and anaplasmosis. We have also investigated cytotoxic effect of IDP on equine Peripheral mononuclear cells (PBMCs) and Vero cell line. The data generated indicate that the IC of IDP (2.73 µM) was significantly lower than its cytotoxic concentration (32.66 and 80.70 µM against PBMCs and Vero cell line, respectively). We have recorded signific...
Blastocyst production by conventional in vitro fertilization (cIVF) in horses: Effects of sperm storage method, incubation timing of cool-stored semen before gamete co-incubation, and comparisons between cIVF and intracytoplasmic sperm injection (ICSI). Currently, Intracytoplasmic Sperm Injection (ICSI) on in vitro-matured equine oocytes obtained by transvaginal oocyte aspiration (TVA) is the method of choice for in vitro production of equine blastocysts in a clinical setting. A protocol for conventional in vitro fertilization (cIVF) using either fresh or frozen/thawed stallion sperm incubated under capacitating conditions (Tyrode's Albumin Lactate Pyruvate medium + penicillamine, hypotaurine, epinephrine; FERT-PHE) has been recently reported. Several factors that may impact the clinical applicability of this cIVF protocol have yet to be st...
Preliminary investigation of the effect of ferulic acid on miRNAs and LncRNAs in Mongolian horse skeletal muscle satellite cells. 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...
Orientin Reverses Premature Senescence in Equine Adipose Stromal Cells Affected by Equine Metabolic Syndrome Through Oxidative Stress Modulation. Equine metabolic syndrome (EMS) is a prevalent endocrine disorder associated with insulin dysregulation, oxidative stress, and impaired regenerative capacity of adipose-derived stem cells (ASCs). The aim of this study was to evaluate the effects of orientin-a plant-derived flavonoid with known antioxidant properties-on equine ASCs (EqASCs) derived from both clinically healthy and diagnosed EMS-affected mares. EqASCs were treated with orientin to evaluate its biological effects. The analysis included key cellular functions such as proliferative capacity, viability, apoptosis, oxidative stress, ...
Comparison of 3.5- and 4.0-mm Cortical Bone Screws for Stabilization of an Equine Navicular Bone Fracture Model. The aim of this study was to compare the strength (load at failure) and the number of cycles to failure of 3.5- and 4.0-mm cortical screw-bone constructs for repair of navicular bone fractures in horses.This was an in vitro biomechanical study that used a four-point bending apparatus to determine the load at failure of 3.5- and 4.0-mm cortical screw-bone constructs subjected to static and cyclic loading.In static testing, pairwise comparison of 4.0-mm cortical screw-bone constructs demonstrated significantly greater strength (mean increase: +16%, = 0.0135) compared with those in the 3.5-m...
Effect of Pre-IVM Duration with cAMP Modulators on the Production of Cloned Equine Embryos and Foals. The asynchrony of cytoplasmic and nuclear maturation in cumulus-oocyte complexes (COCs) due to prematurely declining concentrations of cyclic adenosine monophosphate (cAMP) has been shown to result in reduced oocyte developmental competence. The objective of this study was to evaluate the effect of pre-IVM treatment with cAMP modulators for different durations on the developmental potential of equine oocytes used for cloned embryo production. Collected COCs were transferred to cryovials filled with transport medium at 20-22 °C. Within the cryovials, the COCs were either untreated (Control) fo...
Direct Warming of Vitrified In Vivo Equine Embryos. Vitrified in vitro-produced embryos can be successfully warmed in isotonic media at room temperature (RT; 22°C). However, this protocol has not been reported for in vivo embryos, which are more challenging to vitrify and warm. Study objectives were to see if vitrified in vivo embryos warmed in RT isotonic medium gave equivalent pregnancy rates to stepwise serial dilution warming, and if embryo size influenced the results. One hundred and seventeen embryos were divided into groups by size (G1:≤ 300 μm, n = 59; G2:> 300-400 μm, n = 33; G3:> 400-500 μm, n = ...
Influence of Cryopreservation on the Acrosome Reaction in Hucul Stallion Spermatozoa. The Hucul horse is a Polish primitive breed with a small population size, which highlights the importance of preserving the genetic resources. The cryopreservation of semen is essential for creating gene banks, but its effect on the acrosome reaction in Hucul stallions has not yet been investigated. The acrosome reaction is one of the most important physiological events associated with the fertilization process. Therefore, our goal was to determine the level of acrosome reaction in chilled and frozen/thawed Hucul stallion semen using the FluoAcro test and the SCA semen analysis system. We foun...
Pantoea agglomerans in Equine Ulcerative Keratitis: Prevalence and Comparative Efficacy of Four Topical Antiseptics. To determine the minimal bactericidal concentration (MBC) and effective contact time of four topical antiseptics-polyhexanide, povidone-iodine (PVP-I), hypochlorous acid (HOCl), and N-acetylcysteine (NAC)-against Pantoea agglomerans, a pathogen frequently isolated in equine ulcerative keratitis. Methods: Over a 17-month sampling period, clinical isolates were collected from horses with ulcerative keratitis. The most frequently isolated strain (Pantoea agglomerans, n = 14) was selected for in vitro analysis. Methods: All isolates were used to determine the MBCs of the four antiseptics. Eac...
Follicular fluid secretome enhances the meiotic competence of in vitro matured equine oocytes, but not the developmental competence of zygotes after intracytoplasmic sperm injection (ICSI). In vitro embryo production is widely used in equine clinical reproduction; however, its efficiency remains suboptimal due to the undermined developmental competence of in vitro-matured oocytes. In this work, we evaluated the effect of mare preovulatory follicular fluid (PFF) secretome supplementation during in vitro maturation (IVM) of equine oocytes recovered post-mortem. Secretome was isolated from PFF and was added to the IVM medium at concentrations of 20 µg/ml (S20) or 40 µg/ml (S40). The percentage of mature oocytes, as well as embryo cleavage, blastocyst development, and gene expr...
Mechanical Stimulation of Equine Bone Marrow Mesenchymal Stromal Cell-Derived Cartilage-Like In Vitro Model Triggers Osteoarthritis Features. Osteoarthritis (OA) affects millions of people globally, causing irreversible cartilage damage, chronic inflammation, and progressive joint dysfunction. Similarly, horses can develop OA spontaneously or due to their athletic careers, influenced by mechanical and biochemical factors. Current treatments primarily focus on symptom relief without promoting cartilage regeneration. In line with the 3Rs principles (refine, reduce, replace), the development of OA models is essential for advancing new therapeutic approaches against OA. In response to this need, the present study aimed to develop an m...
Catalase in Unexpected Places: Revisiting H2O2 Detoxification Pathways in Stallion Spermatozoa. Oxidative stress plays a critical role in regulating sperm function, yet species-specific antioxidant mechanisms remain poorly understood. This study compared hydrogen peroxide (H2O2) tolerance in horse and human sperm and investigated the roles of catalase and glutathione peroxidase (GPx) in horses. A H2O2 dose-response assay (0-2000 µM) showed that horse sperm were significantly more resistant to oxidative damage, with an IC50 for progressive motility over 14-fold higher than that of human sperm (391.6 µM vs. 27.3 µM). Horse sperm also accumulated more intracellular H2O2 without loss of m...
Proteomic profiling reveals the potential role of allogenic equine platelet-rich plasma and extracellular vesicles in modulating tendon inflammation and repair. To determine the protein composition of equine platelet-rich plasma (PRP) and PRP-derived extracellular vesicles (EVs) and evaluate their effects on tendon inflammation in vitro. As tendon injuries are common in horses and treatment with PRP derived from the horse's own blood shows promise, but outcomes vary due to inconsistent composition. PRP contains EVs that facilitate cell communication. Unassigned: From December 2022 through May 2023, equine plasma (n = 3, adult) was isolated via double centrifugation and PRP produced using a commercial kit. Extracellular vesicles were isolated using dif...
Preimplantation genetic testing in horses: biopsy of Piezo-ICSI embryos for sex, coat color, and disease alleles. The introduction of ovum pick-up, Intracytoplasmic Sperm Injection (ICSI) and in vitro embryo production in equine reproduction offers significant advantages, including overcoming infertility, high blastocyst yields, embryo cryopreservation, and preimplantation genetic testing (PGT) by collecting few cells extruded spontaneously during embryo growth using a mouth pipette. PGT enables embryo selection before transfer and is particularly useful in breeds with a preference for specific offspring genders, coat color, and detecting genetic diseases. Here, we present the first large-scale validation...
Biphasic CAPA-IVM Improves Equine Oocyte Quality and Subsequent Embryo Development Without Inducing Genetic Aberrations. In vitro maturation (IVM) of oocytes retrieved from ovum pick-up (OPU) or ovarian tissue (OT) is a standard approach for patients with specific conditions where prior hormonal stimulation is contraindicated. However, the developmental competence of oocytes matured in vitro is still inferior to that of oocytes matured in vivo. Capacitation IVM (CAPA-IVM) includes an extra step of pre-maturation culture (PMC) with c-type natriuretic peptide (CNP) as a meiotic arrestor to better synchronize cytoplasmic and nuclear maturity in oocytes by allowing the cytoplasm additional time to acquire essential ...
Culicoides allergens expressed in insect cells induce sulphidoleukotriene release from peripheral blood leukocytes of horses affected with insect bite hypersensitivity. Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis in horses caused by bites of spp. The allergens are salivary gland proteins from these insects, and nine major allergens from have been identified and expressed in . However, proteins expressed in procaryotic systems have limitations in cellular assays, particularly in functional assays assessing the allergen-induced release of mediators , such as sulphidoleukotrienes (sLT) from basophils. The aims of the study were to produce functional allergens in insect cells, to assess their allergenicity using a sLT release assay, and to...
Development of a two-layer 3D equine endometrial tissue model using genipin-crosslinked collagen scaffolds and 3D printing. Advances in endometrial tissue engineering have enabled the combination of modified scaffolding materials with modern cell culture technologies. Genipin and three-dimensional (3D) printing have advanced cell-tissue engineering by enabling the precise layering of cell-containing matrices while ensuring low cytotoxicity. This study aimed to advance equine endometrial tissue engineering by designing customized collagen scaffolds using 3D printing technology, while optimizing the genipin concentration to avoid toxicity. Genipin was tested at concentrations of 4, 2, 1, 0.5, 0.25, 0.125, and 0 mM on...
Does gradient isolation preserve equine neutrophil viability and oxidative function for up to six hours? 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...
Tenogenic potential of tendon-derived mesenchymal stem cells isolated post-mortem: Impact of cryopreservation. In situ injection of mesenchymal stem cells appears as a promising treatment of tendinopathies. Tendon-derived mesenchymal stem cells (TDSCs) are widely studied and show a lot of interesting characteristics for clinical use. The aim of this study is to confirm the tenogenic potential of cryopreserved TDSCs and to confirm their ability to produce type I and/or type III collagens fibers in culture. Tendon-derived mesenchymal stem cells are harvested from the tendon no later than 72 h post-mortem. Their tenogenic potential has been assessed by quantitative reverse transcriptase polymerase chain ...
Impact of hydrolysable and condensed tannin sources and levels on in vitro equine cecal fermentation. Environmental impacts of animal agriculture are under intense scrutiny, thus strategies to decrease greenhouse gas emissions and volatile nitrogen waste are valuable. As dietary inclusion of tannins has been shown to decrease methanogenesis and improve nitrogen retention in ruminants, this study was designed to determine the effects of graded levels of hydrolysable tannin (chestnut) and condensed tannin (mimosa) on equine in vitro cecal fermentation. Methods: Two in vitro experiments were conducted, each with three replications per treatment. Treatments were 0.2 g alfalfa without or with 5, 1...
In vitro model of equine cartilage degradation; using cartilage pellets differentiated from bone marrow-derived mesenchymal stem cells. The self-renewal capacity of chondrocytes in osteoarthritis (OA) joints is limited, and mesenchymal stem cells (MSCs) are crucial in disease treatment. This study established an OA model from equine bone marrow-derived mesenchymal stem cells (eBMSCs). The eBMSCs were cultured and differentiated into chondrocytes to generate cartilage pellets, which were induced for 7 d with inflammatory cytokines, interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) to mimic OA conditions. Treated culture medium was collected to estimate enzyme activity (MMP-2, MMP-3, and MMP-9) using zymograph...
Substrate stiffness modifies gene expression and transcriptional response of equine endometrial fibroblasts to TGF-β1. Equine endometrial fibrosis is a leading cause of subfertility in aging mares. Fibrosis is a reparative response involving excess deposition of extracellular matrix (ECM) and increasing tissue stiffness. Augmented rigidity itself can drive fibrosis, by stimulating transition from fibroblasts to myofibroblasts. Myofibroblasts release latent transforming growth factor beta 1 (TGF-β1) from the ECM, thereby activating this profibrotic cytokine. Tissue culture polystyrene (TCP) is commonly used for in vitro experiments. The endometrium, however, is considerably softer than TCP. This study critical...
The relationship between post-thaw sperm quality parameters and blastocyst production following Intracytoplasmic Sperm Injection (ICSI) of in vitro-matured equine oocytes. In vitro production of equine embryos by Intracytoplasmic Sperm Injection (ICSI) is commonly utilized to maximize the availability of stallion sperm, particularly from stallions with limited semen supply. The relationship between standard measures of sperm quality in frozen/thawed semen (i.e., sperm motility, normal morphology, DNA quality) and embryo production after ICSI of in vitro-matured equine oocytes has not been thoroughly studied. In this study, frozen/thawed semen from 47 stallions utilized in a commercial ICSI program was analyzed for post-thaw sperm total and progressive motility (...
