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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.
Vitrification of Equine In Vivo-Derived Embryos After Blastocoel Aspiration. Embryo cryopreservation is normally performed with great success in species like humans and cattle. The large size of in vivo-derived equine embryos and the presence of a capsule-impermeable to cryoprotectants-have complicated the use of embryo cryopreservation in equine reproduction. A breakthrough for this technique was obtained when large equine embryos could be successfully cryopreserved after collapsing the blastocoel cavity using a micromanipulation system. High pregnancy rates have been obtained when vitrification is used in combination with embryo collapse.
Cryopreservation of Semen from Domestic Livestock: Bovine, Equine, and Porcine Sperm. In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow cooling from room temperature to 5 °C, packaging of insemina...
Effects of L-Carnitine on Equine Semen Quality During Liquid Storage. l-Carnitine (LC) plays a key role in sperm metabolism, easily providing energy through β-oxidation, which positively affects motility. The objective of this study was to investigate the association between blood plasma and seminal plasma LC levels, as well as the effect of LC as an additive in a skimmed milk-based extender during sperm storage at 5°C. In the first experiment, semen and blood samples from 14 Quarter Horse stallions were used. The LC content in blood plasma and seminal plasma was determined by spectrophotometry and their relationships with seminal parameters were evaluated. In...
Single-step purification of equine chorionic gonadotrophin directly from plasma using affinity chromatography. Equine chorionic gonadotrophin (eCG) is a hormone widely used in superovulation protocols because of its follicle-stimulating action, which increases reproductive efficiency in animals of productive interest. It contains 45% carbohydrate, 10% of which is N-acetylneuraminic acid (sialic acid). The eCG purification procedures from equine serum or plasma are mainly based on chromatographic methods. However, before these procedures, it is necessary to follow sample pre-conditioning steps, such as several precipitation stages and/or ultrafiltration/diafiltration processes. In this work, an efficien...
Laboratory transmission potential of British mosquitoes for equine arboviruses. There has been no evidence of transmission of mosquito-borne arboviruses of equine or human health concern to date in the UK. However, in recent years there have been a number of outbreaks of viral diseases spread by vectors in Europe. These events, in conjunction with increasing rates of globalisation and climate change, have led to concern over the future risk of mosquito-borne viral disease outbreaks in northern Europe and have highlighted the importance of being prepared for potential disease outbreaks. Here we assess several UK mosquito species for their potential to transmit arboviruses ...
The P-glycoprotein repertoire of the equine parasitic nematode Parascaris univalens. P-glycoproteins (Pgp) have been proposed as contributors to the widespread macrocyclic lactone (ML) resistance in several nematode species including a major pathogen of foals, Parascaris univalens. Using new and available RNA-seq data, ten different genomic loci encoding Pgps were identified and characterized by transcriptome-guided RT-PCRs and Sanger sequencing. Phylogenetic analysis revealed an ascarid-specific Pgp lineage, Pgp-18, as well as two paralogues of Pgp-11 and Pgp-16. Comparative gene expression analyses in P. univalens and Caenorhabditis elegans show that the intestine is the maj...
Sodium Caseinate and Cholesterol Improve Bad Cooler Stallion Fertility. This study aimed to assess the effects of sodium caseinate and cholesterol to extenders used for stallion semen cooling. Two ejaculates from 19 stallions were extended to 50 million/mL in four different extenders and cooled-stored for 24 hours at 5°C. The extender 1 (E1) consisted of a commercially available skim milk-based extender. The extender 2 (E2) consisted of E1 basic formula with the milk component being replaced by sodium caseinate (20 g/L). The extender 3 (E3) consisted of E1 basic formula added to cholesterol (1.5 mg/120 million sperm). The extender 4 (E4) consisted of a combinat...
Identification and characterization of the enzymes responsible for the metabolism of the non-steroidal anti-inflammatory drugs, flunixin meglumine and phenylbutazone, in horses. The in vivo metabolism and pharmacokinetics of flunixin meglumine and phenylbutazone have been extensively characterized; however, there are no published reports describing the in vitro metabolism, specifically the enzymes responsible for the biotransformation of these compounds in horses. Due to their widespread use and, therefore, increased potential for drug-drug interactions and widespread differences in drug disposition, this study aims to build on the limited current knowledge regarding P450-mediated metabolism in horses. Drugs were incubated with equine liver microsomes and a panel of r...
Effect of MnTBAP on in vitro capacitation of frozen-thawed stallion sperm. In vitro manipulation of spermatozoa leads to deleterious changes of structure and function that occur mainly due to oxidative stress, therefore, prevention or treatment is a strategy to improve the functions of processed sperm. In the present study, the aim was to evaluate the effects of MnTBAP supplementation, a compound with antioxidant activity, on in vitro capacitation conditions of thawed equine sperm. For this purpose, stallion spermatozoa (2 × 10 cells/mL) were incubated in the sperm-TLP base medium for 4 h in which there were three different conditions: non-capacitating, capacitating...
In vitro Effects of Methylprednisolone Acetate on Equine Deep Digital Flexor Tendon-Derived Cells. Primary deep digital flexor tendon (DDFT) pathologies and those accompanying degenerative changes of navicular bone fibrocartilage are major causes of lameness associated with navicular disease. Intrasynovial corticosteroids are mainstay in the treatment due to the anti-inflammatory effects, but their effect on DDFT cell biosynthesis are unknown. The objective of this study was to investigate the effects of methylprednisolone acetate (MPA) on cells isolated from the dorsal fibrocartilaginous region of forelimb DDFTs (DDFT-derived cells) of 5 horses (aged 11-17 years). Non-adherent aggregate c...
Discrepancies in the bilateral intradermal test and serum tests in atopic horses. In equine atopic patients intradermal testing (IDT) and immunoglobulin (Ig)E serology are used frequently. There is little evidence regarding the reproducibility of the IDT and IgE serology in horses. Objective: To compare the results of a simultaneously performed IDT on the left and right side of the neck in atopic horses, and to compare these results with allergen-specific IgE serology. Methods: Ten equine patients from a university hospital population with chronic urticaria and/or pruritus. Methods: The IDT was performed using 16 allergens and the results were evaluated after 30 min, 1, 4 ...
Quality control and immunomodulatory potential for clinical-grade equine bone marrow-derived mesenchymal stromal cells and conditioned medium. This study aimed to assess the safety and reproducibility of cell therapy for its use in clinical practice. We performed immunophenotypic characterization of equine bone marrow-derived mesenchymal stromal cells (BMMSCs) by flow cytometry using CD90, CD19, CD14, CD105, CD45, and HLA-DR markers (n = 4); GTG banding cytogenetic analysis (n = 3); and microbiological quality control (n = 4). The immunomodulatory potentials of BMMSCs (n = 4) and its conditioned medium (CM, n = 3) were investigated by in vitro lymphocyte inhibition assay using phytohemagglutinin (PHA)-stimulated peripheral ...
Effects of insulin on IGF-1 receptors in equine lamellar tissue in vitro. Although it is understood that equine endocrinopathic laminitis can be triggered by high concentrations of insulin, it is unclear whether this represents a direct action on lamellar tissue via insulin receptors (InsR), an interaction with IGF-1 receptors (IGF-1R), or some other, indirect action. This uncertainty is because of the reported scarcity of InsR in lamellar tissue and the low affinity of insulin for equine IGF-1R. In the present study, the effects of insulin and IGF-1 (as a positive control) were examined using lamellar explants isolated from the hooves of healthy horses and incubate...
Effects of pituitary pars intermedia dysfunction and Prascend (pergolide tablets) treatment on endocrine and immune function in horses. It remains unclear how pituitary pars intermedia dysfunction (PPID) and pergolide treatment (Prascend [pergolide tablets]) affect endocrine and immune function in horses. To evaluate these effects, blood was collected regularly from 28 university-owned horses (10 Non-PPID, 9 PPID control [PC], and 9 PPID treatment [PT]) over approximately 15 mo. Pergolide treatment was initiated after Day 0 collections. Analyses included ACTH, insulin, total cortisol, free cortisol, complete blood counts, plasma myeloperoxidase, and cytokine/receptor gene expression in basal whole blood and in vitro stimulat...
Irradiating frozen-thawed stallion sperm with red-light increases their resilience to withstand post-thaw incubation at 38 °C. The aim of this study was to evaluate whether red-light stimulation increases the longevity and resilience of cryopreserved stallion sperm to withstand post-thaw incubation for 120 min. Sixteen frozen straws of 0.5 mL from eight stallions were used. Samples were cryopreserved, thawed through incubation at 38 °C for 30 s and divided into the control and samples exposed to red-light using a triple LED photo-activation system (wavelength: 620-630 nm). Three irradiation protocols consisting of different light-dark-light intervals (1-1-1, 2-2-2 and 3-3-3 min) were tested. Sperm quality param...
Optimizing corneal riboflavin administration in ex vivo horse, dog, rabbit, and pig samples for use in corneal collagen cross-linking. Determine optimal iontophoresis times for riboflavin delivery to the corneal stroma across different species and compare these to corneal injection. Methods: Ex vivo horse, dog, rabbit, and pig globes were treated with riboflavin administered with either iontophoresis for 2.5-20 minutes with or without corneal epithelium; or with purpose-designed precise corneal injection (PCI) application with intact epithelium. Immediately following riboflavin administration, samples were harvested, frozen, and sectioned. Riboflavin penetration was imaged using fluorescence microscopy. Results: Horse sample...
Publisher Correction: Differences in the intrinsic chondrogenic potential of equine umbilical cord matrix and cord blood mesenchymal stromal/stem cells for cartilage regeneration. An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Puncture of the Equine Embryonic Capsule and Its Repair In Vivo and In Vitro. Vitrification of embryos >300 µm in diameter requires puncture of the glycoprotein capsule, although the size of the hole compatible with embryo survival is unknown. Forty-five day-7 or -8 embryos were punctured using a 30-µm glass biopsy pipette mounted on a micromanipulator (n = 20) or manually with either an acupuncture needle (∼100-µm diameter -hole; n = 10) or a microneedle with a <1 µm tip to produce a ∼30-µm diameter hole (n = 15) before transferring to recipient mares; further 12 embryos were punctured with either the acupuncture needle or microneedle before being cul...
Growth Rates of Thoroughbred Foals and In Vitro Gut Health Parameters When Fed a Cereal or an All-Fiber Creep Feed. Traditions and the economic advantage when producing big athletic Thoroughbred (TB) yearlings for sale encourage owners to feed high levels of cereals. Such diets can predispose gastric ulceration and developmental orthopedic diseases, impacting negatively on future careers. This study aimed to (1) determine if an all-fiber creep feed (total mixed fiber ration [TMFR] would sustain equal growth rates in TB foals versus a cereal-based stud cube (SC) and (2) compare pH and lactate produced by SC and TMFR in in vitro foregut and hindgut conditions. Eight TB foals in matched-pairs were fed SC or T...
Gram-positive bacteriocins: usage as antimicrobial agents in veterinary medicine. Antimicrobial resistance is a worldwide spread phenomenon that affects both human and veterinary medicine. This issue has led to a "One Health" approach in order to coordinate efforts and set back the development of drug-resistant microbes. In the search for alternatives therapies, bacteriocins or antimicrobial peptides have proven to be effective both in vitro and in vivo for multiples pathogens, even those resistant to many classic antibiotics. Gram-positive bacteriocins have been the most studied to the present. The use of bacteriocins as therapeutically active molecules is limited mainly d...
Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole. Parascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years, Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways. Methods: Adult worms were collected from the intestines of two foals at slaughter. The foals were natu...
Endothelial cells and angiogenesis in the horse in health and disease-A review. The cardiovascular system is the first functional organ in the embryo, and its blood vessels form a widespread conductive network within the organism. Blood vessels develop de novo, by the differentiation of endothelial progenitor cells (vasculogenesis) or by angiogenesis, which is the formation of new blood vessels from existing ones. This review presents an overview of the current knowledge on physiological and pathological angiogenesis in the horse including studies on equine endothelial cells. Principal study fields in equine angiogenesis research were identified: equine endothelial progen...
Equine herpesvirus type 1 affects mitochondrial network morphology and reactive oxygen species generation in equine dermal cell line. In the present study, the influence of the infection with equine herpesvirus type 1 (non-neuro-pathogenic and neuropathogenic strains of EHV-1) on the morphology and distribution of mitochondrial network in equine dermal cell line was investigated. Our results indicate that EHV-1-infection caused changes in the mitochondrial morphology manifested mostly by fission and reactive oxygen species generation.
Use of in vitro assays to identify antibiotics that are cytotoxic to normal equine chondrocytes and synovial cells. Intra-articular (IA) antibiotic usage is prevalent in equine practice. However, recent emergence of antimicrobial resistance prompts re-evaluation of antibiotic selection, particularly when used prophylactically. Furthermore, many commonly used antibiotics exert direct cytotoxicity to equine cells, and appropriate IA doses have not been defined. Objective: To screen antibiotics in vitro as an initial assessment of cytotoxicity against normal equine joint cells in monolayer culture and explant tissues. Methods: In vitro experimental study. Methods: Chondrocytes and synovial cells were harvested...
Harvesting, processing, and evaluation of in vitro-manipulated equine preantral follicles: A review. The mammalian ovary is responsible for essential stages of folliculogenesis and hormonal production, regulating the female physiological functions during the menstrual/estrous cycles. The mare has been considered an attractive model for comparative studies due to the striking similarities shared with women regarding in vivo and in vitro folliculogenesis. The ovarian follicular population in horses contains a large number of oocytes enclosed in preantral follicles that are yet to be explored. Therefore, the in vitro manipulation of equine preantral follicles aims to avoid the process of atre...
A composite hydrogel-3D printed thermoplast osteochondral anchor as example for a zonal approach to cartilage repair: in vivo performance in a long-term equine model. Recent research has been focusing on the generation of living personalized osteochondral constructs for joint repair. Native articular cartilage has a zonal structure, which is not reflected in current constructs and which may be a cause of the frequent failure of these repair attempts. Therefore, we investigated the performance of a composite implant that further reflects the zonal distribution of cellular component both in vitro and in vivo in a long-term equine model. Constructs constituted of a 3D-printed poly(ϵ-caprolactone) (PCL) bone anchor from which reinforcing fibers protruded into ...
Supportive techniques to investigate in vitro culture and cryopreservation efficiencies of equine ovarian tissue: A review. During the reproductive lifespan of a female, only a limited quantity of oocytes are naturally ovulated; therefore, the mammalian ovary possesses a substantial population of preantral follicles available to be handled and explored in vitro. Hence, the manipulation of preantral follicles enclosed in ovarian tissue aims to recover a considerable population of oocytes of high-value animals for potential application in profitable assisted reproductive technologies (ARTs). For this purpose, the technique of preantral follicle in vitro culture (IVC) has been the most common research tool, achievin...
Effects of cysteine and ascorbic acid in freezing extender on sperm characteristics and level of enzymes in post-thawed stallion semen. Present study aimed to investigate the effect of adding antioxidants, cysteine and ascorbic acid on the levels of glutamic oxaloacetic transaminase (GOT), glutamic-pyruvate (GPT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and γ-glutamyl transpeptidase (GGT) enzymes of post-thawed stallion sperm. Ten ejaculates were collected each from four healthy stallions and cryopreserved using HF-20 freezing extender containing either 0 mg/ml cysteine or ascorbic acid, 0.5 mg/ml cysteine and 0.5 mg/ml ascorbic acid. All samples in freezing extender containing cysteine or ascorbic acid or ...
Histomorphometric and vascular changes in equine endometrium after the infusion of conceptus fragments. This experiment aimed to verify if the proteins present in a 13 day conceptus induce changes in the equine endometrial ultra-structure, histology, and vascularization, two days after its infusion. Ten healthy cyclic mares were used. Once estrus was confirmed, mares were examined daily to detect ovulation (day 0). After ovulation, mares were examined daily until day seven by transrectal palpation and B-mode and Doppler ultrasonography. In this first cycle, intrauterine biopsies were collected at day seven after ovulation, constituting the Cyclic group (n = 10). In the second cycle, the same mar...
Effects of Autologous Conditioned Serum, Autologous Protein Solution, and Triamcinolone on Inflammatory and Catabolic Gene Expression in Equine Cartilage and Synovial Explants Treated With IL-1β in Co-culture. Autologous conditioned serum (ACS) and autologous protein solution (APS) are newer therapeutic options for osteoarthritis (OA). Co-culture of cartilage and synovium stimulated with IL-1β produces a similar physiologic response to tissues from naturally-ocurring OA. The study objective was to investigate the effects of ACS, APS, and triamcinolone (TA) on inflammatory and catabolic gene expression of inflamed joint tissues in co-culture. Blood was collected and processed for ACS and APS from six horses. Cartilage and synovial explants were harvested from the stifle, placed in co-culture, and tr...
