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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.
Interleukins decrease epithelial protein and gene expression by K14 + CD105+ equine hoof progenitor cells. Background: Progenitor cell epithelial to mesenchymal transition (EMT) could impact healing in the epidermal-dermal junction of the equine hoof stratum internum (SI). The hypothesis of this study was that K14 + CD105+ equine hoof SI progenitor cells assume a mesenchymal phenotype in the presence of inflammatory interleukins in vitro.Methods: K14 + CD105+ progenitor cell percentages isolated from proliferative scarred (fibrous) and healthy SI were quantified. Decellularized matrix was prepared from and K14 + CD105+ cells localized in healthy SI. Gene expression (CD44, CD105, E-cadhe...
Terpenic compounds possess anthelmintic and immunomodulatory properties with potential for controlling equine cyathostomin infections. The emergence of anthelmintic-resistant parasite isolates necessitates alternative control strategies. The use of dietary additives with anti-parasitic or immunomodulatory activities has been proposed as a solution, but whether such additives can simultaneously exert dual bioactivities within this context has rarely been explored. Here, we evaluated whether selected terpenes could exert dual bioactivities (anti-parasitic and immunomodulatory) with a view to their use as food additives that can limit strongyle infections in horses. In vitro tests with cyathostomin larval development, larval mig...
Milk fat globule EGF and factor V/VIII domain containing (MFGE8) as a novel player in equine endometrial fibrosis. With increasing age, mares are frequently affected by endometrial fibrosis. Transforming growth factor beta 1 (TGF-β1) promotes fibrosis, while milk fat globule EGF and factor V/VIII domain containing (MFGE8) primarily exerts antifibrotic effects. We used in situ hybridization analysis (ISH) to investigate MFGE8, TGFB1 and its downstream effectors cellular communication network factor 2 (CCN2) and transgelin (TAGLN) in equine endometrial fibrosis. Additionally, transcriptional effects of TGF-β1 and MFGE8 on equine endometrial fibroblasts in vitro were investigated by single cell sequencing. ...
In Vitro Characterization of an Equinized Anti-PD-L1 Antibody for Cancer Immunotherapy in Horses. Horses develop spontaneous tumors, typically in old age. Although local tumor control can be achieved using conventional therapies, systemic therapies are required to treat recurrent and/or metastatic tumors. Immune checkpoint inhibitors, such as anti-PD-L1 antibodies, have been approved for the treatment of various tumor types in humans; however, little is known about the immunosuppressive roles of the PD-1/PD-L1 pathway in horses, and the therapeutic potential of these inhibitors remains to be elucidated. Previously, we reported that the rat monoclonal anti-PD-L1 antibody 6C11-3A11 cross-rea...
Mass culture of equine synovial fluid-derived mesenchymal stromal cells using nonwoven polyethylene terephthalate fabrics. Culture protocols need to yield 100 million equine synovial fluid (SF)-derived mesenchymal stromal cells (SF-MSCs) in around 3 weeks are needed, before these cells can be evaluated as agents of articular repair in clinical trials. Objective: To investigate mass culture of equine SF-MSC culture protocols using nonwoven polyethylene terephthalate (PET) fabrics for the potential to meet the targets for clinical trials. Methods: In vitro experiments. Methods: SF samples were collected from the carpal joints in thoroughbred racehorses (n = 21) undergoing arthroscopic surgery and cultured in 10%...
Impact of donor mare and stallion identity on the likelihood of pregnancy after transfer of in vitro produced embryos. The success of in vitro embryo production (IVEP) is influenced by donor mare and stallion. Objective: To determine whether donor mare and stallion influence the pregnancy rate after transfer of in vitro produced (IVP) blastocysts and to identify factors influencing the likelihood of obtaining one or more pregnancies from a single IVEP session. Methods: Retrospective clinical study. Methods: The influence of the following variables on pregnancy outcome was assessed in 2292 transfers of IVP blastocysts: speed of in vitro embryo development, embryo grade after thawing, age of the donor mare, blas...
Serum-free medium modulates the immunomodulatory and anabolic function of equine bone marrow-derived mesenchymal stromal cells. Adult bone marrow-derived mesenchymal stromal cells (BM-MSCs) hold significant therapeutic potential in human and veterinary medicine. Traditionally, BM-MSC cultures use fetal bovine serum (FBS) to promote growth, but this practice introduces batch variability and xenogeneic contamination that complicate clinical translation. While serum-free medium (SFM) alternatives have been optimized for human MSCs, limited options have been explored for alternative or veterinary species. Here, we compared a chemically defined, customizable SFM formulation with conventional serum-containing medium (SCM) fo...
An investigation into the impact of the inclusion of a commercial fiber-based feed, designed for both hydration and nutrition, on the equine hindgut microbiomes. It is more evident that horse health is linked to the gastrointestinal tract and microbial community structure (MCS) as affected by diet. There are many marketed feeds available to enhance nutrition for horses, however the effects of these feeds on hindgut MCS are largely unknown. Objective: The aim was to test the effect of a complementary forage feed (FF) on the fermentation profile and MCS in contrasting basal diets. Methods: A 2×2 factorial design was used to assess the effect of basal diet, forage only (FOR) or forage/concentrate (MIX), and presence/absence of FF on fermentation paramete...
Clinical pregnancy outcomes after transfer of fresh and vitrified-warmed conventional IVF equine embryos. In vitro fertilization (IVF) in horses has emerged as a valuable assisted reproductive technology, yet the impact of embryo cryopreservation on pregnancy outcomes when utilizing this technique remains unclear. This study reports pregnancy outcomes following transfer of conventional equine IVF embryos, with both fresh and vitrified-warmed embryos. Conventional IVF (cIVF) was used to produce 40 embryos, which were transferred to recipient mares, pregnancy diagnosis was performed at 6 days post-transfer followed by embryonic heartbeat detection between day 25 and 30 of gestation. Observed pregnan...
Intra-articular hydrogel therapeutics exhibit anti-inflammatory effects on cytokine-stimulated equine cartilage and synovial membrane explants. To investigate whether IA hydrogel therapeutics alter the inflammatory and/or degradative responses of articular cartilage (AC) and synovial membrane (SM) to cytokine stimulation. Unassigned: AC and SM tissue explants were stimulated with interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α) and simultaneously treated with triamcinolone acetonide, hyaluronic acid, collagen-elastin hydrogel microparticles (CEHM), or 2.5% polyacrylamide hydrogel (2.5% iPAAG) at 3 doses. Synovial membrane (n = 9 explants, 3 horses) was stimulated/treated for 18 hours, whereas AC (12 explants, 6 horses) wa...
Effect of Acer species (red and Freeman maple) and horse characteristics, management, and location on the in vitro oxidation of equine erythrocytes. Red maple toxicosis arises when horses consume wilted maple leaves, resulting in methemoglobinemia and hemolytic anemia due to oxidative damage to erythrocytes. Objective: The objective of this study was to determine how maple species (red versus Freeman maple) and horse characteristics, management, and barn site contributed to in vitro methemoglobin production and hemolysis. Methods: Horse information was obtained through an owner questionnaire. Extracts from red and Freeman maple leaves were separately incubated with washed erythrocytes from each horse (n=120). The in vitro percentage methem...
Synergistic effects of Ferula asafoetida extract and condensed tannins from raisin pomace on in vitro cecal fermentation kinetics and nutrient digestibility in horses. The equine hindgut depends on microbial fermentation for efficient nutrient utilization but remains vulnerable to dysbiosis, hindgut acidosis, and suboptimal fiber digestion. Growing restrictions on antibiotic and synthetic feed additives have increased interest in natural phytogenic compounds. Medicinal plant extracts and condensed tannins are promising candidates to modulate microbial activity, improve fermentation efficiency, and enhance nutrient digestibility. This study aimed to investigate the individual and combined effects of hydroalcoholic extract of and condensed tannins extracted f...
Are antioxidants hindering stallion sperm capacitation? A perspective on hidden barriers to equine IVF success. 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.
Characterization of isolated late preantral and early antral equine ovarian follicles. 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...
Temporal characterization of conditions that promote functional capacitation of stallion sperm. Equine in vitro fertilization (IVF) has historically been challenged due to lack of conditions that promote functional capacitation of stallion sperm. Capacitation success is often attributed to prolonged sperm incubation requirements. Advances to equine IVF are hindered by incomplete characterization of conditions that promote the functional capacitation of stallion sperm over time. Herein, we describe conditions that promote the functional capacitation of fresh, cooled, and frozen-thawed stallion sperm isolated by Percoll, Swim-Up, or Microfluidics (VetMotl). Fresh (diluted sperm) and previo...
Cannabidiol-induced cellular and matrix-associated responses in primary equine sarcoid cells. 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 ...
Antibiofilm and resistance-modulating properties of tt-farnesol against Streptococcus equi subsp. equi and zooepidemicus. The aim of this study was to investigate the antibacterial, antibiofilm and modulating activities of trans-trans-farnesol (tt-farnesol) on two strains of Streptococcus equi subsp. equi and two strains of S. equi subsp. zooepidemicus, pathogenic or commensal bacteria of horses. Tt-farnesol showed bactericidal activity against all tested strains, with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) between 8 and 4 μg/mL. The sesquiterpene caused significant damage to the bacterial membrane, and its antibacterial activity did not appear to depend on oxidat...
Cannabidiol-rich extract suppresses the activation of proinflammatory genes IL-1β and IL-6 in equine mesenchymal stem cells stimulated with lipopolysaccharide. Peripheral nerve injuries (PNI) often lead to long-term functional impairment. Mesenchymal stem cells (MSCs) and cannabidiol (CBD) have shown anti-inflammatory and neuroprotective effects in vitro, which may be relevant for PNI research. The aim of this study was to evaluate CBD-rich cannabis extract’s potential to induce anti-inflammatory and neurotrophic gene expression in equine adipose tissue-derived MSCs (EqAT-MSCs) in an inflammatory in vitro environment. The morphology and metabolic activity of EqAT-MSCs (n = 4) were assessed after CBD-rich extract priming at concentrations of 3, ...
Metabolomics reveals early predictors of blastocyst formation in equine ICSI-derived embryos. 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...
In vitro anthelmintic activity of extracts and bioactive compounds from Azadirachta indica and Cnidoscolus angustidens on strongyles L3 larvae in horses. 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...
Antiparasitic potential of Caesalpinia coriaria: Current evidence and prospects for equine health. 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...
Nicotinic acid treatment improves the developmental potential of equine oocytes for cloned embryo production. 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...
Cell and Gene Therapy in Equine Ocular Disease. 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...
Generation and characterization of an immortalized equine osteoblast cell line. 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...
Tafenoquine succinate inhibits the growth of the equine piroplasmosis hemoparasites Theileria equi and Babesia caballi. Equine piroplasmosis (EP) is a tick-borne disease of equids caused by the intraerythrocytic apicomplexan parasites Theileria equi, Babesia caballi and the recently identified Theileria haneyi. Acute cases can be severe, with anemia, jaundice, abortion or sudden death. Survivors remain lifelong carriers, serving as reservoirs for tick-borne and iatrogenic transmission. No vaccines are currently available, and control strategies rely heavily on accurate diagnostics and chemotherapeutic intervention. Imidocarb dipropionate (ID) is the current standard of care for both acute treatment and radical ...
Polyhexanide, Povidone-Iodine, and Hypochlorous Acid Show High In Vitro Antimicrobial Efficacy Against Pathogens Commonly Associated With Equine Infectious Keratitis. To determine the in vitro antimicrobial activity of specific antiseptics against common equine ocular surface pathogens. Methods: Staphylococcus aureus (S. aureus) (n = 12), Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) (n = 9), Enterobacter hormaechei (E. hormaechei) (n = 6), and Bacillus cereus (B. cereus) (n = 5) were collected from corneal samples of horses with ulcerative keratitis. Reference strains were included. Minimum bactericidal concentrations (MBC) of polyhexanide, povidone-iodine, and hypochlorous acid were tested using the microdilution method. ...
Functional and molecular characterization of equine intestinal organoids across media conditions and intestinal segments. Gastrointestinal (GI) disease is a major cause of morbidity and mortality in horses, with disruption of the intestinal epithelial barrier playing a central role in disease pathogenesis. A deeper understanding of the molecular and functional properties of the equine intestinal barrier is essential to improve diagnostics and therapeutics. While intestinal organoids have emerged as a promising tool for modeling GI physiology and disease, equine-specific data remain limited. Existing studies vary in methodology and often lack functional characterization, particularly across different intestinal re...
Assessing the Sperm Head DNA Damage in Frozen/Thawed Horse Spermatozoa via Xenogeneic ICSI. In the mouse, spermatozoa are highly resistant to DNA damage, even when frozen without cryoprotectants, and can produce offspring when subsequently used for ICSI (intracytoplasmic sperm injection). It is not known whether the same applies to other mammals as well. For example, in the horse, even conventional sperm freezing is still very problematic and frequently leads to sperm immobility. It has, however, never been tested whether sperm immobility also mirrors sperm head DNA damage, and if so, to what extent. In our study, we evaluated the damage to DNA in horse frozen and thawed motile and i...
Impact of melatonin on inflammatory cytokine profiles in 24-hour cultured equine uterine explants’. Melatonin has demonstrated beneficial effects in regulating inflammatory processes in various tissues, but its role in mare uterine physiology remains unclear. Objective: To investigate the effects of melatonin on the gene expression of cytokines (IL-1β, IL-6, IL-8, IL-10, and TNF-α) in equine uterine explants cultured for 6, 12, or 24 hours. Methods: The study was divided into two experiments. Part 1 aimed to evaluate the cytotoxicity of melatonin at concentrations of 0, 0.5, 1, 2, 4, and 8 mM on endometrial cells. Part 2 aimed to determine the expression of inflammatory cytokines in uterin...
Anti-Theileria equi activity of methanolic extract of Artemisia scoparia: In vitro efficacy, in vivo safety, and identification of lead molecules. Equine piroplasmosis is a tick-borne disease that affects horses and is caused by Theileria equi and Babesia caballi. Currently, imidocarb dipropionate is the primary treatment for T. equi infections; however, its application is limited by its potential systemic toxicity. Artemisia annua is widely recognised for its antimalarial properties, whereas Artemisia scoparia has traditionally been used for various medicinal purposes. However, the therapeutic potential of A. scoparia against theileriosis in animals remains largely unknown. Activity-guided fractionation of a methanolic extract of A. sco...
