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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.
Nucleotide sequence of complementary DNA encoding for quaking protein of cow, horse and pig. Complementary DNA (cDNA) for bovine quaking gene (Bqk), equine quaking gene (Eqk) and porcine quaking gene (Pqk), which are homologous to mouse quaking gene (qkI), were isolated, and their nucleotide sequences were determined. cDNA sequences of Bqk, Eqk and Pqk showed very high homology to that of qkI at nucleotide level; 94.2, 95.7 and 95.6%, respectively. Deduced amino acid sequences for Bqk, Eqk and Pqk perfectly matched to that of qkI. These findings suggest that the quaking gene family is highly conserved during mammalian evolution, and that Bqk, Eqk and Pqk are likely to have important b...
The sperm chromatin structure assay: a review of clinical applications. The sperm chromatin structure assay (SCSA) was introduced by as a method to determine the susceptibility of sperm DNA to denaturation and how those results related to fertility. This initial study used human sperm and was followed by studies in bulls and boars . This assay was one of the first to introduce the technique of flow cytometry, which has the ability to evaluate specific sperm compartments of large numbers of sperm in a short time, as a methodology to evaluate sperm quality and further define the relationship of sperm quality to fertility. For any assay to be of use clinically, it mu...
Comparative chromosomal studies of E. caballus (ECA) and E. przewalskii (EPR) in a female F1 hybrid. Previous research revealed that the karyotypes of Equus przewalskii (2n = 66) and Equus caballus (2n = 64) differ by one pair of metacentric chromosomes, present in ECA but not in EPR, and two pairs of acrocentric chromosomes found only in the EPR karyotype. The formation of a trivalent during meiosis in a male F1 hybrid and the homologies in G-banding patterns suggest that ECA 5 corresponds to two acrocentric EPR chromosomes resulting from a Robertsonian fusion or fission event. Chromosomal investigations of a female interspecies F1 hybrid including banded karyograms and fluorescence in situ ...
Mechanical and functional properties of the equine superficial digital flexor tendon. The in vitro and in vivo mechanical properties of the superficial digital flexor tendon have been described. To date the focus has been on single load to failure testing, however refined in vivo methods may prove useful to evaluate the effects of treatment and exercise on tendons. During maximal exercise, the adult superficial digital flexor tendon operates close to its functional limits with a narrow biomechanical safety margin. This combined with exercise and age associated microdamage, and a limited adaptive ability may increase the risk of fatigue failure. Studies evaluating treatment regi...
The role of stallion seminal proteins in fertilisation. Seminal plasma proteins are secretory proteins originating mainly from the epididymis and the accessory sex glands. They are involved in the remodelling of the sperm surface which occurs during sperm transit through the male genital tract and continues later at ejaculation. During this process, collectively called post-testicular sperm maturation, the spermatozoa acquire the ability to fertilise an egg. Seminal plasma proteins have been shown to contribute to early and central steps of the fertilisation sequence, e.g. the establishment of the oviductal sperm reservoir, modulation of capacitati...
Effects of recombinant equine growth hormone on in vitro biomechanical properties of the superficial digital flexor tendon of Standardbred yearlings in training. To determine whether recombinant equine growth hormone (rEGH) would alter the in vitro biomechanical properties of the forelimb superficial digital flexor tendon (SDFT) in exercising young Standardbred horses. Methods: Randomized complete block design. Methods: Twelve Standardbred yearlings. Methods: Horses were trained for 12 weeks on a high-speed treadmill (10% positive incline). rEGH was administered intramuscularly (IM) daily (10 microg/kg during week 4; 20 microg/kg for weeks 5-9) to 6 horses (treated group), whereas 6 horses (control group) were administered an equivalent daily volume of...
Assessment of stiffness and strength of 4 different implants available for equine fracture treatment: a study on a 20 degrees oblique long-bone fracture model using a bone substitute. To compare the mechanical properties of 4 stabilization methods for equine long-bone fractures: dynamic compression plate (DCP), limited contact-DCPlate (LC-DCP), locking compression plate (LCP), and the clamp-rod internal fixator (CRIF--formerly VetFix). Methods: In vitro mechanical study. Methods: Bone substitute material (24 tubes) was cut at 20 degrees to the long axis of the tube to simulate an oblique mid-shaft fracture. Methods: Tubes were divided into 4 groups (n=6) and double plated in an orthogonal configuration, with 1 screw of 1 implant being inserted in lag fashion through the "fr...
Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival. Cryopreservation induces partially irreversible damage to equine sperm membranes. Part of this damage occurs due to membrane alterations induced by the membrane changing from the fluid to the gel-state as the temperature is reduced lower than the membrane transition temperature. One way to prevent this damage is to increase the membrane fluidity at low temperatures by adding cholesterol to the membrane. Different concentrations of cholesterol-loaded-cyclodextrins (CLC) were added to stallion sperm to determine the CLC concentration that optimizes cryosurvival. Higher percentages of motile sper...
Characterization of the in vitro responses of equine cecal longitudinal smooth muscle to endothelin-1. To characterize the in vitro response of equine cecal longitudinal smooth muscle (CLSM) to endothelin (ET)-1 and assess the role of ETA and ETB receptors in those ET-1-induced responses. Methods: 36 horses without gastrointestinal tract disease. Methods: To determine cumulative concentration-response relationships, CLSM strips were suspended in tissue baths containing graded concentrations of ET-1 (10(-9) to 10(-6)M) with or without BQ-123 (ETA receptor antagonist); with or without IRL-1038 (ETB receptor antagonist); or with both antagonists at concentrations of 10(-9), 10(-7), and 10(-5)M. To...
Molecular cloning and characterization of markers and cytokines for equid myeloid cells. The myeloid cell system comprises of monocytes, macrophages (MPhi), dendritic cells (DC), Kupffer cells, osteoclasts or microglia and is also known as the mononuclear phagocytic system (MPS). Essential cytokines to differentiate or activate these cells include GM-CSF or IL-4. Important markers for characterization include CD1, CD14, CD68, CD163 and CD206. All these markers, however, were not cloned or further characterized in equids by use of monoclonal antibodies earlier. To overcome this problem with the present study, two approaches were used. First, we cloned equine cytokines and markers, ...
Components in seminal plasma regulating sperm transport and elimination. Seminal plasma has been suggested to be involved in sperm transport, and as a modulator of sperm-induced inflammation, which is thought to be an important part of sperm elimination from the female reproductive tract. This article reports on recent experiments on the importance of seminal plasma components in sperm transport and elimination. In Experiment 1, hysteroscopic insemination in the presence (n = 3) or absence (n = 3) of 2 ng/mL PGE showed an increased portion of spermatozoa crossing the utero-tubal junction in the presence of PGE in two mares, while no difference was observed between ...
Amides as cryoprotectants for freezing stallion semen: a review. Stallion semen cryopreservation, despite its impact on the horse industry, is not an established technology. During the last years, a number of modifications have been proposed to the freezing process, however, a large population of stallions still have poor semen quality and fertility after frozen-thawed. Glycerol toxicity could be a reason for the variation on stallion sperm freezability. There are limited publications concerning the use of alternative cryoprotectants for equine sperm. Glycerol is contraceptive for some species and other cryoprotectors, such as amides, have been show to be a...
Equine FISH mapping of 36 genes known to locate on human chromosome ends. The INRA and the CHORI-241 horse BAC libraries were screened by hybridization with DNA probes and/or directly by PCR with primers designed in consensus sequences of genes localized at the end of each human chromosome. BAC clones were retrieved and 36 could be FISH mapped after the expected gene was confirmed in each BAC by sequencing. Our results show that 16 BACs can be considered to be at telomeric or centromeric positions in the horse and 15 were found at the boundary of actually defined conserved segments even-though often located within conserved syntenic fragments between horse and human...
Equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze-thaw cycles. This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract...
Microvascularization and angiogenic activity of equine corpora lutea throughout the estrous cycle. Corpus luteum growth and endocrine function are closely dependent on the formation of new capillaries. The objectives of this study were to evaluate (i) tissue growth and microvascular development in the equine cyclic luteal structures; (ii) in vitro angiogenic activity of luteal tissues in response to luteotrophic (LH, PGE(2)) and luteolytic (PGF(2alpha)) hormones and (iii) to relate data to luteal endocrinological function. Our results show that microvascular density was increased in the early and mid luteal phase, followed by a fall in the late luteal phase and a further decrease in the cor...
Integration of future biotechnologies into the equine industry. There has and will continue to be reproductive techniques available that have a positive impact upon the equine breeding industry. This review focuses on semen technologies that have been developed or are in the process of being developed. The use of fluorescent dyes and flow cytometry has provided the researcher and clinician with powerful tools to evaluate several sperm attributes. These procedures have been utilized to evaluate sperm viability, acrosome status, mitochondrial status, DNA integrity and stages of capacitation. Flow cytometry allows several sperm attributes to be evaluated on t...
Factors affecting the plasma membrane function of cooled-stored stallion spermatozoa. The spermatozoon is a highly specified cell that has the abilities of active motility and fertilization of the ovum. Damage to the sperm plasma membrane results in the irreversible loss of its functions. Because of the high content of unsaturated fatty acids in the plasma membrane, mammalian sperm are sensitive to oxidative stress. While mild peroxidation appears to promote capacitation of the sperm cell, excessive peroxidation will damage the plasma membrane and results in loss of motility and fertility. The functional integrity of the sperm plasma membrane can be determined by functional tes...
Separation and detection of the isomeric equine conjugated estrogens, equilin sulfate and delta8,9-dehydroestrone sulfate, by liquid chromatography–electrospray-mass spectrometry using carbon-coated zirconia and porous graphitic carbon stationary phases. Equilin-3-sulfate and delta8,9-dehydroestrone-3-sulfate are two isomers found in equine conjugated estrogens that differ in structure only by the position of a double bond in the steroid B-ring. These geometric isomers were not resolved on a C18 column during the analysis of conjugated estrogen drug products by LC-MS using acetonitrile-ammonium acetate buffer as the mobile phase. While no separations of these two isomers were observed on C18 or other alkyl-bonded silica based phases using a variety of mobile phase conditions, partial separations were achieved on phenyl bonded silica phases wit...
Capacitation, acrosome function and chromatin structure in stallion sperm. In general, fertility in breeding stallions is lower and more variable than in the other farm animal species, primarily because selection is based on pedigree, looks and/or athletic performance, with little consideration of fertility or fertility potential. Moreover, because the average stallion breeds only a limited number of mares per year and in-field fertility is influenced significantly by non-stallion factors such as management and mare fertility, meaningful fertility data are hard to come-by. Unfortunately, generating usable figures would involve impractically high costs, time and numbe...
Sequence analysis of canine and equine ferritin H and L subunit cDNAs. Canine and equine ferritin H and L subunit cDNA clones were obtained using reverse transcriptase-polymerase chain reaction (RT-PCR) and TA cloning from various tissues. Canine liver and spleen ferritin H subunit cDNA clones contained an open reading frame for the same 182-amino acid protein as that reported in canine brain ferritin H subunit cDNA although there were substitutions in the 3'-noncoding regions. Ferritin L subunit cDNA clones from canine liver, spleen, and kidney showed identical coding sequences encoding the 174-amino acid protein except for a single nucleotide substitution in ki...
In vitro evaluation of the effect of the opioid antagonist N-methylnaltrexone on motility of the equine jejunum and pelvic flexure. Although potent analgesics, opioids decrease intestinal activity, leading to ileus in many species. N-methylnaltrexone (MNTX), an opioid antagonist which does not cross the blood-brain barrier and antagonises the morphine effect on the intestine, directly stimulates motility and restores function without affecting analgesic properties. While its use has been reported in human subjects, there is no information with regard to its usage in the horse. Objective: To determine whether MNTX has an effect on contractile activity of the equine jejunum and pelvic flexure. Methods: Using circular smooth ...
Structure of myelin P2 protein from equine spinal cord. Equine P2 protein has been isolated from horse spinal cord and its structure determined to 2.1 A. Since equine myelin is a viable alternative to bovine tissue for large-scale preparations, characterization of the proteins from equine spinal cord myelin has been initiated. There is an unusually high amount of P2 protein in equine CNS myelin compared with other species. The structure was determined by molecular replacement and subsequently refined to an R value of 0.187 (Rfree=0.233). The structure contains a molecule of the detergent LDAO and HEPES buffer in the binding cavity and is otherwise ...
Isolation of equine herpesvirus-1 lacking glycoprotein C from a dead neonatal foal in Japan. We isolated a variant equine herpesvirus-1 (EHV-1), strain 5089, from the lung of a dead neonatal foal in Japan and characterized the biological nature of the virus. The virus spread in cultured cells mainly by cell-to-cell infection, unlike wild-type EHV-1, which spreads efficiently as a cell-free virus. The virus titer in cultured supernatant and the intracellular virus titer were low compared to those of wild-type EHV-1. Heparin treatment of the virus had no effect on viral infectivity in cell culture. Glycoprotein C (gC) was not detected by Western blotting and fluorescent antibody tests i...
The development and application of the modern reproductive technologies to horse breeding. Although the horse was probably the first animal to experience and benefit from artificial insemination, it trailed the field somewhat with regard to the application of embryo transfer and other oocyte and embryo-related modern breeding technologies. But with a late run it is now back in mid-field and gaining fast on the other large domestic species in the application of the many technological advances of the past 20 years to sound breeding practice. Improvements in extenders and cryoprotectants have resulted in a veritable upsurge in the transport and insemination of cooled and frozen stallio...
In vitro evaluation of the contractile response to endothelin-1 of the circular and longitudinal myometrial layers of the uterine horn of nongravid mares. To characterize the in vitro response of circular and longitudinal myometrial layers of the uterine horn (CMLH and LMLH, respectively) of horses to endothelin (ET)-1 by use of specific ETA (BQ-123) and ETB (IRL-1038) receptor antagonists. Methods: Uteruses from 10 nongravid mares in anestrus. Methods: Muscle strips from the CMLH and LMLH were suspended in tissue baths and connected to force-displacement transducers interfaced with a polygraph. Strips were incubated for 45-minute intervals with no antagonist (control specimens), and 3 concentrations (10(-9), 10(-7), and 10(-5)M) of BQ-123, IRL-...
Characterization of an A-kinase anchor protein in equine spermatozoa and examination of the effect of semen cooling and cryopreservation on the binding of that protein to the regulatory subunit of protein kinase-A. To determine whether a homologue of A-kinase anchor protein 4 (AKAP4) is present and functional as an AKAP in equine spermatozoa and examine the effect of semen cooling and cryopreservation on binding of equine AKAP4 to the regulatory (RII) subunit of protein kinase-A (PK-A). Methods: Ejaculated semen collected from 2 fertile stallions, 3 bulls, and 3 humans. Methods: Identification of an equine homologue of AKAP4 was investigated via DNA sequencing. Protein was extracted from the spermatozoa of each species for immunoblot analysis to identify AKAP4 and its precursor protein, pro-AKAP4; immuno...
Effect of caspase inhibitors on the post-thaw motility, and integrity of acrosome and plasma membrane of cryopreserved equine spermatozoa. The present study was designed to test the hypothesis that addition of anticaspase cocktails (inhibiting caspases and thus blocking apoptosis) to the extenders increases the post-thaw viability of equine spermatozoa. The addition of caspase inhibitors failed to improve the acrosome and plasma membrane integrity of spermatozoa, suggesting that in equine sperm cryopreservation protocols, the addition of these caspase inhibitors to cryopreservation medium may not be beneficial in protecting the sperm from the stress of cryopreservation.
Equine infectious anemia virus Gag p9 function in early steps of virus infection and provirus production. We have previously reported that serial truncation of the Gag p9 protein of equine infectious anemia virus (EIAV) revealed a progressive loss in replication phenotypes in transfected cells, such that a proviral mutant (E32) expressing the N-terminal 31 amino acids of p9 produced infectious virus particles similarly to parental provirus, while a proviral mutant (K30) with two fewer amino acids produced replication-defective virus particles, despite containing apparently normal levels of processed Gag and Pol proteins (C. Chen, F. Li, and R. C. Montelaro, J. Virol. 75:9762-9760, 2001). Based on ...
An in vitro study on spontaneous myometrial contractility in the mare during estrus and diestrus. Uterine smooth muscle specimens were collected from euthanatized mares in estrus and diestrus. Longitudinal and circular specimens were mounted in organ baths and the signals transcribed to a Grass polygraph. After equilibration time and 2 g preload, their physiologic isometric contractility was recorded for a continuous 2.0 h. Area under the curve, frequency and time occupied by contractions were studied. Differences between cycle phases, between muscle layers, and over the recorded time periods were statistically evaluated using linear mixed-effect models. In the mare, physiologic contractil...
