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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.
Investigating the impact of extracellular vesicle addition during IVM on the fertilization rate of equine oocytes following ICSI. The efficacy of in vitro embryo production (IVEP) in equines is relatively limited compared to other species due to the lack of a reliable superovulation technique, limited availability of cumulus oocyte complexes (COCs), low in vitro oocyte maturation (IVM) and fertilization rates. Extracellular vesicles (EVs), which are nanoparticles involved in intercellular signaling in the ovarian environment, have shown potential as supplements to improve oocyte development during IVM. This study tested the hypothesis that EVs from small (< 20 mm) ovarian follicles could enhance fertilization rates i...
IVF with frozen-thawed sperm after prolonged capacitation yields comparable results to ICSI in horses: A morphokinetics study. Intracytoplasmic sperm injection (ICSI) is the current clinical practice for the in vitro production of equine embryos. The use of conventional fertilization methods such as in vitro fertilization (IVF), has historically been associated with poor success in horses. However, recent improvements have led to better outcomes with IVF, though only when using fresh semen, which limits its use in clinical practice. IVF remains in its infancy in equine reproduction, and several unknowns remain about the technique. One significant gap in knowledge concerns the morphokinetics of IVF embryos and how they...
Matrikine stimulation of equine synovial fibroblasts and chondrocytes results in an in vitro osteoarthritis phenotype. Osteoarthritis (OA) is a debilitating disease that impacts millions of individuals and has limited therapeutic options. A significant hindrance to therapeutic discovery is the lack of in vitro OA models that translate reliably to in vivo preclinical animal models. An alternative to traditional inflammatory cytokine models is the matrikine stimulation model, in which fragments of matrix proteins naturally found in OA tissues and synovial fluid, are used to stimulate cells of the joint. The objective of this study was to determine if matrikine stimulation of equine synovial fibroblasts and chond...
Equine bone marrow-derived mesenchymal stromal cells reduce established S. aureus and E. coli biofilm matrix in vitro. Biofilms reduce antibiotic efficacy and lead to complications and mortality in human and equine patients with orthopedic infections. Equine bone marrow-derived mesenchymal stromal cells (MSC) kill planktonic bacteria and prevent biofilm formation, but their ability to disrupt established orthopedic biofilms is unknown. Our objective was to evaluate the ability of MSC to reduce established S. aureus or E. coli biofilms in vitro. We hypothesized that MSC would reduce biofilm matrix and colony-forming units (CFU) compared to no treatment and that MSC combined with the antibiotic, amikacin sulfate...
Assessment of equine intestinal epithelial junctional complexes and barrier permeability using a monolayer culture system. Gastrointestinal disease is a leading cause of death in mature horses. A lack of in vitro modeling has impeded the development of novel therapeutics. The objectives of this study were to develop and further characterize a small intestinal monolayer cell culture derived from equine jejunum including establishing normal measurements of intestinal permeability and restitution. Three-dimensional enteroids, derived from postmortem sampling of equine jejunum, were utilized to develop confluent epithelial monolayers. The presence of differentiated intestinal epithelial cell types and tight junctions ...
In vitro antimicrobial activity of non-traditional therapies for infectious endometritis in mares. Endometritis is the leading cause of subfertility in horses, and it is a clinical problem where historically antibiotics have not always been used with prudent justification. Because of this, alternative therapies to treat endometritis are necessary for the rational use of antibiotics. Objective: To assess the in vitro antimicrobial activity of non-traditional therapies commonly used in clinical practice against microorganisms causing infectious endometritis in mares. Methods: In vitro experiments. Methods: A microdilution technique was performed to determine the minimum inhibitory concentrati...
Sex hormone-binding globulin promotes the osteogenic differentiation potential of equine adipose-derived stromal cells by activating the BMP signaling pathway. Musculoskeletal injuries and chronic degenerative diseases pose significant challenges in equine health, impacting performance and overall well-being. Sex Hormone-Binding Globulin (SHBG) is a glycoprotein determining the bioavailability of sex hormones in the bloodstream, and exerting critical metabolic functions, thus impacting the homeostasis of many tissues including the bone. Unassigned: In this study, we investigated the potential role of SHBG in promoting osteogenesis and its underlying mechanisms in a model of equine adipose-derived stromal cells (ASCs). An SHBG-knocked down model has b...
A new hormonal protocol supports early development of in vitro-produced embryos after transfer to anoestrus mares. The present study aimed to evaluate whether primed anoestrus mares are suitable recipients for embryos produced by intracytoplasmic sperm injection (ICSI). Anoestrus was confirmed in four mares and daily doses of oestradiol benzoate (6 mg in total) over 5 days were administered; after 3 days of rest, oral altrenogest was administered at 0.088 mg/kg and embryos (1 to 5 embryos per mare; 15 in total) were transferred 3.5 days after progesterone onset. Uterine lavage was conducted 48 h after transfer. The results revealed an 80% embryo recovery rate, and among the retrieved embryos, 6...
Prolonged incubation of frozen-thawed equine spermatozoa for in vitro fertilization: A preliminary study using low temperature and INRA96 medium. A protocol for conventional in vitro fertilization (IVF) in horses using fresh semen has been described, using a prolonged incubation in FERT-TALP medium (22 h) at 38.2°C in the presence of penicillamine, hypotaurine and epinephrine (PHE). Our work aimed to develop a protocol that maintains quality parameters in frozen-thawed equine spermatozoa incubated for 22 h in the presence of PHE using different media (FERT-TALP and INRA96) and incubation temperatures (30 and 38.2°C). Twelve frozen ejaculates from four stallions were thawed and then incubated in either FERT-TALP or INRA96 with PHE...
Equine coronavirus infection and replication in equine intestinal enteroids. In this study, equine intestinal enteroids (EIEs) were generated from the duodenum, jejunum, and ileum and inoculated with equine coronavirus (ECoV) to investigate their suitability as in vitro models with which to study ECoV infection. Immunohistochemistry revealed that the EIEs were composed of various cell types expressed in vivo in the intestinal epithelium. Quantitative reverse-transcription PCR (qRT-PCR) and virus titration showed that ECoV had infected and replicated in the EIEs. These results were corroborated by electron microscopy. This study suggests that EIEs can be novel in vitro ...
Effect of refreezing extender on stallion sperm quality and embryo production after intracytoplasmic sperm injection. Intracytoplasmic sperm injection (ICSI) is a valuable assisted reproduction technology in clinical practice, especially when semen availability is limited. Since the number of sperm required per ICSI cycle is much less than the number of sperm available in a standard straw of frozen semen, refreezing semen at lower sperm concentrations could yield multiple straws for ICSI use. However, there is little data on the effect of sperm refreezing on ICSI outcomes, especially on the effect of extender used for refreezing. The objective of the present study was to evaluate the effect of refreezing exte...
In vitro characterization of radiofrequency ablation lesions in equine and swine myocardial tissue. Radiofrequency ablation is a promising technique for arrhythmia treatment in horses. Due to the thicker myocardial wall and higher blood flow in horses, it is unknown if conventional radiofrequency settings used in human medicine can be extrapolated to horses. The study aim is to describe the effect of ablation settings on lesion dimensions in equine myocardium. To study species dependent effects, results were compared to swine myocardium. Right ventricular and right and left atrial equine myocardium and right ventricular swine myocardium were suspended in a bath with circulating isotonic sali...
Isolation-protocol, characterization, and in-vitro performance of equine umbilical vein endothelial cells. Angiogenesis plays a crucial role in various physiological and pathological conditions. However, research in equine angiogenesis is relative limited, necessitating the development of suitable models. To effectively analyze angiogenesis in-vitro, it is essential to target the specific cells responsible for this process, namely endothelial cells. Human umbilical vein endothelial cells (HUVECs) are one of the most used models for studying angiogenesis in humans. Serving as an equivalent to HUVECs, we present a comprehensive isolation protocol for equine umbilical vein endothelial cells (EqUVECs...
Investigating the Suitability of Mare’s Milk-Derived Exosomes as Potential Drug Carriers. Exosomes are cell-derived, membrane-surrounded particles that deliver bioactive molecules to various cells. Due to their small size, low immunogenicity, extended blood circulation, and involvement in cellular communication, they hold potential as effective drug carriers. Exosomes are present in various biological fluids, including mare's milk, a traditional drink in Central Asia. This study aims to compare exosome isolation methodologies and determine the stability of mare's milk-derived exosomes as potential therapeutic carriers. Three extraction methods-immunoprecipitation, size exclusion ch...
Characterization of Nasal Mucosal T Cells in Horses and Their Response to Equine Herpesvirus Type 1. Equine herpesvirus type 1 (EHV-1) enters through the upper respiratory tract (URT). Mucosal immunity at the URT is crucial in limiting viral infection and morbidity. Here, intranasal immune cells were collected from horses (n = 15) during an experimental EHV-1 infection. CD4+ and CD8+ T cells were the major intranasal cell populations before infection and increased significantly by day six and fourteen post-infection, respectively. Nasal mucosal T cells were further characterized in healthy horses. Compared to peripheral blood mononuclear cells (PBMC), mucosal CD8+ T-cell percentages were elev...
Equine metabolic investigation of the phosphodiesterase-4 inhibitor ibudilast as a potential performance enhancer. Phosphodiesterase 4 (PDE4) inhibitors are a newer class of drugs that induce bronchodilation and have anti-inflammatory effects, making them susceptible to misuse as performance enhancers in competitive sports. Methods: This study explores the metabolic conversion of PDE4 inhibitor ibudilast in thoroughbred horses after oral administration and in vitro using equine liver microsomes and Cunninghamella elegans. A liquid chromatography-high resolution mass spectrometry method was used to postulate the plausible structures of the detected metabolites. Results: A total of 20 in vivo metabolites wer...
Effect of protein acetylation on capacitation of stallion sperm. Sperm capacitation is considered the main factor limiting conventional in vitro fertilization (IVF) in horses. A recent scientific breakthrough in sperm processing for IVF in horses has resulted in embryos and foals being produced; however, various aspects of the IVF process remain to be fully elucidated. Lysine acetylation has been shown to play a role in sperm capacitation in several species and the objective of this study was to detect and evaluate this process in the horse. Ejaculates of two stallions were collected and incubated in different conditions with deacetylase inhibitors to induc...
Maturational competence of equine oocytes is associated with alterations in their ‘cumulome’. Assisted reproductive technologies are an emerging field in equine reproduction, with species-dependent peculiarities, such as the low success rate of conventional IVF. Here, the 'cumulome' was related to the developmental capacity of its corresponding oocyte. Cumulus-oocyte complexes collected from slaughterhouse ovaries were individually matured, fertilized by ICSI, and cultured. After maturation, the cumulus was collected for proteomics analysis using label-free mass spectrometry (MS)-based protein profiling by nano-HPLC MS/MS and metabolomics analysis by UPLC-nanoESI MS. Overall, a total o...
Pre- and Post-Thaw Addition of L-Carnitine and Pyruvate: Effect on Stallion Sperm Parameters. 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...
Revealing the Therapeutic Potential of Muscle-Derived Mesenchymal Stem/Stromal Cells: An In Vitro Model for Equine Laminitis Based on Activated Neutrophils, Anoxia-Reoxygenation, and Myeloperoxidase. Laminitis in horses is a crippling condition marked by the deterioration of the dermal-epidermal interface, leading to intense lameness and discomfort, often necessitating euthanasia. This study aimed to establish an in vitro model of laminitis using a continuous keratinocyte cell line exposed to anoxia-reoxygenation and an activated neutrophil supernatant. A significant decrease in the keratinocytes' metabolism was noted during the reoxygenation period, indicative of cellular stress. Adding muscle-derived mesenchymal stem/stromal cells during the reoxygenation demonstrated a protective effect...
Assessing the effects of ex vivo hormonal exposure on oxidative responses in equine leukocytes: A preliminary study. Breed differences exist between horses and ponies in circulating concentrations of several hormones, notably ACTH and insulin. These hormones regulate stress and metabolic responses, but in other species, they also impact leukocyte oxidant responses. The effects of these hormones on equine leukocytes have not been evaluated to date. If equine leukocytes are similarly regulated, breed differences in increased plasma hormone concentrations or altered sensitivity to them at the leukocyte level could result in breed-related differences in oxidant responses or oxidative status. The objective of thi...
Proteome and metabolomic profile of Mongolian horse follicular fluid during follicle development. During follicular development, changes in the composition of the follicular fluid are synchronized with the development of oocytes. Our aim was to screen the key factors affecting oocyte maturation and optimize the in vitro culture protocol by understanding the changes of proteins and metabolites in follicular fluid. Follicles are divided into three groups according to their diameter (small follicle fluid (SFF): 10 mm < d < 20 mm; medium follicle fluid (MFF): 20 mm < d < 30 mm; large follicle fluid (LFF): 30 mm < d). Proteins and metabolites from the fol...
Studies in vitro of equine intestinal glucagon-like peptide-2 secretion. Equine insulin dysregulation (ID) is a significant metabolic problem because the hyperinsulinaemia that develops increases the animal's risk of developing laminitis, a debilitating foot condition. The role of gastrointestinal factors, such as incretin hormones, in the pathogenesis of ID and hyperinsulinaemia in horses is poorly understood, particularly in comparison to other species. Glucagon-like peptide-2 (GLP-2) is an intestinotrophic peptide released from L cells in the gastrointestinal tract and is implicated in metabolic dysfunction in other species. The aim of this study in vitro was to...
A SIRT1-independent mechanism mediates protection against steroid-induced senescence by resveralogues in equine tenocytes. Tendinopathy is a common age-related disease which causes significant morbidity for both human athletes and performance horses. In the latter, the superficial digital flexor tendon is an excellent model for human tendinopathies because it is a functional homologue of the human Achilles tendon and a primary site of injuries with strong similarities to the human disease. Corticosteroids have been previously used clinically to treat tendinopathic inflammation, but they upregulate the p53-p21 axis with concomitant reductions in cell proliferation and collagen synthesis in human tenocytes. This phe...
In vitro, the aging of stallion spermatozoa at 22 °C is linked to alteration in Ca2+ and redox homeostasis and may be slowed by regulating metabolism. Conservation of equine semen in the liquid state is a central procedure in horse breeding and constitutes the basis of associated reproductive technologies. The intense mitochondrial activity of the stallion spermatozoa increases oxidative stress along the storage period, leading to sperm demise within 24-48 h of storage, particularly when maintained at room temperature. Recently, the relationship between metabolism and oxidative stress has been revealed. The study aimed to extend the period of conservation of equine semen, at room temperature through modification of the metabolites present i...
Equine uterine sanitizer: in vitro inhibition of endometritis-causing microorganisms and its stability to dilute antibiotics. 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...
Storage of equine faecal microbiota transplantation solution has minimal impact on major bacterial communities and structure. Management of diarrhoea in horses is usually non-specific and supportive. Faecal microbiota transplantations (FMT) are used to manage dysbiosis in horses with diarrhoea. There are few studies investigating the effects of storage on prepared FMT solutions. This study was an in vitro non-randomised controlled experiment that investigated the effects of FMT solution preparation and storage on the faecal microbiota. Fresh faeces were collected from five healthy adult horses and used for DNA extraction and preparation of FMT. From each FMT, seven aliquots were collected and DNA was extracted immedi...
Impact of different formulations of platelet lysate on proliferative and immune profile of equine mesenchymal stromal cells. Platelet lysate (PL) is investigated as a potential replacement for fetal bovine serum (FBS) in cell culture. However, there is limited research on its impact on the immune profile of equine mesenchymal stromal cells (eMSCs). This study aimed to evaluate the effects of different PL formulations on the proliferative capacity, multipotentiality, and immune profile of equine adipose tissue-derived MSCs (eAD-MSCs). growth kinetics and trilineage differentiation of eAD-MSCs ( = 7) were assessed under three culture conditions: medium-concentration PL (MPL), high-concentration PL (HPL), and FBS ...
Livestock embryonic stem cells for reproductive biotechniques and genetic improvement. Embryonic stem cells (ESCs) have proven to be a great model that faithfully recapitulates the events that occur during embryogenesis, making them a unique tool to study the cellular and molecular mechanisms that define tissue specification during embryonic development. Livestock ESCs are particularly attractive and have broad prospects including drug selection and human disease modeling, improvement of reproductive biotechniques and agriculture-related applications such as production of genetically modified animals. While mice and human ESCs have been established many years ago, no significa...
