Topic:Biotechnology
Biotechnology in horses encompasses the application of biological techniques and tools to enhance equine health, performance, and reproduction. This field includes genetic engineering, cloning, and the development of vaccines and therapeutics tailored to equine physiology. Techniques such as gene editing and stem cell therapy are explored for their potential to address genetic disorders, improve tissue regeneration, and enhance disease resistance in horses. Additionally, advancements in reproductive biotechnology, such as artificial insemination and embryo transfer, contribute to genetic diversity and breeding efficiency. This page compiles peer-reviewed research studies and scholarly articles that investigate the applications, methodologies, and implications of biotechnology in equine science.
Advances in sperm cryopreservation in farm animals: Cattle, horse, pig and sheep. Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. In some farm animals, the use of cryopreserved sperm has so many benefits for which relevance has become more evident in recent decades. Values for post-thaw sperm quality, however, are variable among species and within individuals of the same species. There is no standardized methodology for each of the stages of the cryopreservation procedure (andrological examination, semen collection, dilution, centrifugation, resuspension of the pellet with the freezing medium, p...
Purification, molecular characterization and ligand binding properties of the major donkey seminal plasma protein DSP-1. Fibronectin type-II (FnII) family proteins are the major proteins in many mammalian species including bull, horse and pig. In the present study, a major FnII protein has been identified and isolated from donkey (Equus hemionus) seminal plasma, which we refer to as Donkey Seminal Plasma protein-1 (DSP-1). The amino acid sequence determined by mass spectrometry and computational modeling studies revealed that DSP-1 is homologous to other mammalian seminal plasma proteins, including bovine PDC-109 (also known as BSP-A1/A2) and equine HSP-1/2. High-resolution LC-MS analysis indicated that the prot...
Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules. Covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X-ray, NMR, or cryogenic electron microscopy (Cryo-EM), used for protein structure determination. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resolution to be monitored, there is still high demand for alternative, less complicated, and inexpensive approaches. Here, we introduce a new covalent labeling method based on fast fluoroalkylation of proteins (FFAP). FFAP u...
Long-Term Evaluation of Poly(lactic acid) (PLA) Implants in a Horse: An Experimental Pilot Study. In horses, there is an increasing interest in developing long-lasting drug formulations, with biopolymers as viable carrier alternatives in addition to their use as scaffolds, suture threads, screws, pins, and plates for orthopedic surgeries. This communication focuses on the prolonged biocompatibility and biodegradation of PLA, prepared by hot pressing at 180 °C. Six samples were implanted subcutaneously on the lateral surface of the neck of one horse. The polymers remained implanted for 24 to 57 weeks. Physical examination, plasma fibrinogen, and the mechanical nociceptive threshold (MNT) w...
Extracellular vesicles from equine mesenchymal stem cells decrease inflammation markers in chondrocytes in vitro. Mesenchymal stem cells (MSCs) have been used therapeutically in equine medicine. MSCs release extracellular vesicles (EVs), which affect cell processes by inhibiting cell apoptosis and regulating inflammation. To date, little is known about equine EVs and their regenerative properties. Objective: To characterise equine MSC-derived extracellular vesicles (EVs) and evaluate their effect on equine chondrocytes treated with pro-inflammatory cytokines in vitro. Methods: In vitro experiments with randomised complete block design. Methods: Mesenchymal stem cells from bone marrow, adipose tissue, and ...
Evaluation of growth, viability, and structural integrity of equine endometrial organoids following cryopreservation. Reproductive diseases in mares are a significant cause of subfertility and profound economic loss in the equine industry. Utilizing a 3D in vitro cell culture system that recapitulates the in vivo physiology will reduce time, cost, and welfare concerns associated with in vivo reproductive research in mares. If this 3D model is combined with effective cryopreservation, reproductive research on mares can occur year-round, which is not currently possible in this seasonal species. Endometrial organoids, 3D in vitro cell clusters that exhibit in vivo uterine physiology, have been established in mic...
Highly sensitive in vitro bioassay for luteinizing hormone and chorionic gonadotropin allowing their measurement in plasma. In previous studies, we had shown the synergistic effect of 10 M forskolin (FSK) on the detection threshold of the cyclic AMP response to luteinizing hormones (LH) and chorionic gonadotropins (CG) from various species in the mouse Leydig tumor cell (mLTC) cell line. Independently, we started to study the effect of 10-10 M oxytocin (OXT) also on the cyclic AMP response to LH and CG preparations on these same cells and found an amplifying effect on the luminescence response caused by gonadotropins. The aim was then to explore the effects of 10-10 M OXT on the gonadotropin-induced cAMP response, ...
The anti-inflammatory effects of equine bone marrow stem cell-derived extracellular vesicles on autologous chondrocytes. Osteoarthritis (OA) in the horse is an economic and welfare issue and there are no current disease modifying drugs available. Stem cells have been suggested as a therapeutic intervention for OA, originally on the basis of their regenerative capacity. However, it is hypothesised that mesenchymal stem cells (MSC) exert their effects via paracrine factors including the production of extracellular vesicles that can themselves recapitulate the MSC effects in the joint. Objective: To isolate extracellular vesicles from bone marrow MSC and investigate their anti-inflammatory effects on chondrocytes. ...
Towards increasing stallion sperm longevity by storage at subzero temperatures in the absence of ice. The aim of cell preservation technologies is to slow down damaging reactions by lowering the storage temperature. Upon dilution in a stabilizing extender, stallion sperm can be stored at refrigerator temperatures for several days. Cryopreservation allows storage for decades, but freezing and thawing cause damage and viability losses. It is assumed that by storing cells at subzero temperatures in a non-frozen supercooled state, the damaging effects of ice formation can be avoided. In this study, we have investigated if stallion sperm can be stored at -10°C in the absence of ice, and compared v...
INRA96 Supplemented With Phospholipids Liposomes, A Promising Approach for Stallion Sperm Chilling. Among biotechnologies of reproduction in the equine species, artificial insemination remains the most used technology especially for cooled transported sperm. Although the use of INRA96 extender has demonstrated its efficiency for long-term sperm storage at 4°C or 15°C, some stallions ("bad coolers") are excluded from such technology. Some years ago, we demonstrated that liposomes produced from egg yolk (EY) phospholipids could be an alternative to egg yolk plasma in stallion freezing extenders. To develop a new extender for sperm chilling, we evaluated the protective effect of liposomes pr...
Comparative tribology II-Measurable biphasic tissue properties have predictable impacts on cartilage rehydration and lubricity. Healthy articular cartilage supports load bearing and frictional properties unmatched among biological tissues and man-made bearing materials. Balancing fluid exudation and recovery under loaded and articulated conditions is essential to the tissue's biological and mechanical longevity. Our prior tribological investigations, which leveraged the convergent stationary contact area (cSCA) configuration, revealed that sliding alone can modulate cartilage interstitial fluid pressurization and the recovery and maintenance of lubrication under load through a mechanism termed 'tribological rehydration...
New Alternative Mixtures of Cryoprotectants for Equine Immature Oocyte Vitrification. Equine oocyte vitrification would benefit the growing in vitro embryo production programs, but further optimization of the protocol is necessary to reach clinical efficiency. Therefore, we aimed to perform a direct comparison of non-permeating and permeating cryoprotective agents (CPAs) during the vitrification and warming of equine immature oocytes. In the first experiment, cumulus oocytes complexes (COCs) were vitrified comparing sucrose, trehalose, and galactose in combination with ethylene glycol (EG) and dimethyl sulfoxide (DMSO). In the second experiment, the COCs were vitrified using th...
Development of homology model, docking protocol and Machine-Learning based scoring functions for identification of Equus caballus’s butyrylcholinesterase inhibitors. Machine learning (ML), an emerging field in drug design, has the potential to predict toxicity, shape-based analysis of inhibitors, scoring function (SF) etc. In the present study, a homology model, docking protocol, and a dedicated SF have been developed to identify the inhibitors of horse butyrylcholinesterase (BChE) enzyme. Horse BChE enzyme has homology with human BChE and is a substitute for the screening of inhibitors. The developed homology model was validated and the active site residues were identified from Cavityplus to generate grid box for docking. The validation of docking invol...
History repeats itself: horse originated hyperimmune sera production against SARS CoV-2. SARS-CoV-2 disease was announced as a pandemic by The World Health Organization in early 2020. It is still threatening the world population. Here, we aimed to produce hyperimmune sera that contain immunoglobulin G and F(ab')2 fragments sourced from horse antibodies as an urgent response to the pandemic. SARS-CoV-2 was produced and inactivated with three different methods [formaldehyde (FA), formaldehyde, and binary ethylene amine (FA + BEI), and heat treatment]. After in vitro inactivation control, immunogens were mixed with Freund’s adjuvant, thereafter horses (n: 2 for FA, 4 for FA + BEI, ...
Investigation of an injectable gold nanoparticle extracellular matrix. Post-traumatic osteoarthritis (PTOA) is a progressive articular degenerative disease that degrades articular cartilage and stimulates apoptosis in chondrocyte cells. An injectable decellularized, extracellular matrix (ECM) scaffold, that might be able to combat the effects of PTOA, was developed where the ECM was conjugated with 20 nm gold nanoparticles (AuNP) and supplemented with curcumin and hyaluronic acid (HA). Porcine diaphragm ECM was decellularized and homogenized; AuNPs were conjugated using chemical crosslinking followed by mixing with curcumin and/or HA. Injection force testing and...
Pain assessment in horses using automatic facial expression recognition through deep learning-based modeling. The aim of this study was to develop and evaluate a machine vision algorithm to assess the pain level in horses, using an automatic computational classifier based on the Horse Grimace Scale (HGS) and trained by machine learning method. The use of the Horse Grimace Scale is dependent on a human observer, who most of the time does not have availability to evaluate the animal for long periods and must also be well trained in order to apply the evaluation system correctly. In addition, even with adequate training, the presence of an unknown person near an animal in pain can result in behavioral ch...
Low-copy transgene detection using nested digital polymerase chain reaction for gene-doping control. Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of (1) preamplification using...
Performance and automation of ancient DNA capture with RNA hyRAD probes. DNA hybridization-capture techniques allow researchers to focus their sequencing efforts on preselected genomic regions. This feature is especially useful when analysing ancient DNA (aDNA) extracts, which are often dominated by exogenous environmental sources. Here, we assessed, for the first time, the performance of hyRAD as an inexpensive and design-free alternative to commercial capture protocols to obtain authentic aDNA data from osseous remains. HyRAD relies on double enzymatic restriction of fresh DNA extracts to produce RNA probes that cover only a fraction of the genome and can serve a...
Genome-wide scans for signatures of selection in Mangalarga Marchador horses using high-throughput SNP genotyping. The detection of signatures of selection in genomic regions provides insights into the evolutionary process, enabling discoveries regarding complex phenotypic traits. In this research, we focused on identifying genomic regions affected by different selection pressures, mainly highlighting the recent positive selection, as well as understanding the candidate genes and functional pathways associated with the signatures of selection in the Mangalarga Marchador genome. Besides, we seek to direct the discussion about genes and traits of importance in this breed, especially traits related to the typ...
Rho/ROCK Inhibition Promotes TGF-β3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells. Mesenchymal stromal cells (MSC) represent a promising therapeutic tool for tendon regeneration. Their tenogenic differentiation is crucial for tissue engineering approaches and may support their beneficial effects after cell transplantation . The transforming growth factor (TGF)-, signalling via intracellular Smad molecules, is a potent paracrine mediator of tenogenic induction. Moreover, scaffold topography or tendon matrix components induced tenogenesis via activation of the Rho/ROCK cascade, which, however, is also involved in pathological adaptations in extracellular matrix pathologies. Th...
Loading equine oocytes with cryoprotective agents captured with a finite element method model. Cryopreservation can be used to store equine oocytes for extended periods so that they can be used in artificial reproduction technologies at a desired time point. It requires use of cryoprotective agents (CPAs) to protect the oocytes against freezing injury. The intracellular introduction of CPAs, however, may cause irreversible osmotic damage. The response of cells exposed to CPA solutions is governed by the permeability of the cellular membrane towards water and the CPAs. In this study, a mathematical mass transport model describing the permeation of water and CPAs across an oocyte membrane...
Development of a biologically immortalized equine stem cell line. Bone repair in horses implies invasive surgeries and increased cost. Research on musculoskeletal disorders therapy in horses includes cell-based therapy with mesenchymal stromal cells (MSCs). Mesenchymal stromal cells can be obtained from bone marrow (BMMSCs). Unfortunately, BMMSCs have limited cell replication . The objective of this study was to develop a biologically immortalized equine stem cell line derived from bone marrow, with unlimited proliferation and the ability to differentiate into bone cells. Equine BMMSCs were transfected and immortalized with human telomerase reverse transcri...
16 Years of breed management brings substantial improvement in population genetics of the endangered Cleveland Bay Horse. The consequences of poor breed management and inbreeding can range from gradual declines in individual productivity to more serious fertility and mortality concerns. However, many small and closed groups, as well as larger unmanaged populations, are plagued by genetic regression, often due to a dearth in breeding support tools which are accessible and easy to use in supporting decision-making. To address this, we have developed a population management tool (BCAS, Breed Conservation and Management System) based on individual relatedness assessed using pedigree-based kinship, which offers breedi...
Bi-Level Positive Airway Pressure for Non-invasive Respiratory Support of Foals. Respiratory insufficiency and pulmonary health are important considerations in equine neonatal care. As the majority of foals are bred for athletic pursuits, strategies for respiratory support of compromised foals are of particular importance. The administration of supplementary oxygen is readily implemented in equine practice settings, but does not address respiratory insufficiency due to inadequate ventilation and is no longer considered optimal care for hypoxia in critical care settings. Non-invasive ventilatory strategies including continuous or bi-level positive airway pressure are effect...
A Novel QTL and a Candidate Gene Are Associated with the Progressive Motility of Franches-Montagnes Stallion Spermatozoa after Thaw. The use of frozen-thawed semen is an important reproduction tool to preserve the biodiversity of small, native horse breeds such as the Franches-Montagnes (FM). However, not all stallions produce cryotolerant semen with a progressive motility after thaw ≥ 35%. To improve our understanding of the genetic background of male fertility traits in both fresh and frozen-thawed semen, we performed genome-wide association studies (GWAS) on gel-free volume, sperm cell concentration, total sperm count, and progressive motility in fresh and frozen-thawed semen from 109 FM stallions using 335,494 genome-...
Effective protein extraction combined with data independent acquisition analysis reveals a comprehensive and quantifiable insight into the proteomes of articular cartilage and subchondral bone. The objectives of this study was to establish a sensitive and reproducible method to map the cartilage and subchondral bone proteomes in quantitative terms, and mine the proteomes for proteins of particular interest in the pathogenesis of osteoarthritis (OA). The horse was used as a model animal. Protein was extracted from articular cartilage and subchondral bone samples from three horses in triplicate by pressure cycling technology or ultrasonication. Digested proteins were analysed by data independent acquisition based mass spectrometry. Data was processed using a pre-established spectral li...
Allele-specific expression analysis reveals conserved and unique features of preimplantation development in equine ICSI embryos†. Embryonic genome activation and dosage compensation are major genetic events in early development. Combined analysis of single embryo RNA-seq data and parental genome sequencing was used to evaluate parental contributions to early development and investigate X-chromosome dynamics. In addition, we evaluated dimorphism in gene expression between male and female embryos. Evaluation of parent-specific gene expression revealed a minor increase in paternal expression at the 4-cell stage that increased at the 8-cell stage. We also detected eight genes with allelic expression bias that may have an imp...
Sequence determination of phosphorothioated oligonucleotides using MALDI-TOF mass spectrometry for controlling gene doping in equestrian sports. In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). As a model of therapeutic oligonucleotides, 22 bp-long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short-length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 10 (standard deviation: ...
HOX Gene Expressions in Cultured Articular and Nasal Equine Chondrocytes. Osteoarthritis the quality and span of life in horses. Previous studies focused on nasal cartilage as a possible source for autologous chondrocyte implantation (ACI) in cartilage defects in humans. "HOX gene-negative" nasal chondrocytes adapted articular HOX patterns after implantation into caprine joint defects and produced cartilage matrix proteins. We compared the HOX gene profile of equine chondrocytes of nasal septum, anterior and posterior fetlock to identify nasal cartilage as a potential source for ACI in horses. Cartilage was harvested from seven horses after death and derived chondro...