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.
Reubel GH, Studdert MJ.Based on sequence homology with human adenovirus 2 (HAdV2), the hexon gene of equine adenovirus 1 (EAdV1) was identified. HindIII restriction fragments containing the hexon and other viral genes were cloned into the plasmids pUC19 and pBlueScript SK(-) and sequenced. The nucleotide sequence of the hexon gene was completely determined and partial sequence data were obtained for seven other EAdV1 genes. Amino acid (aa) sequence comparison with published adenovirus (AdV) proteins identified the genes for the IIIa, penton, pVII, PVI, 23K proteinase, DNA binding and 100K proteins. The eight EAdV1 g...
Mulligan RS, Livesey JH, Evans MJ, Ellis MJ, Donald RA.It has been suggested that atrial natriuretic peptide (ANP) is the long-sought inhibitor of corticotropin (ACTH) secretion, but the evidence is conflicting. We have examined the effect of ANP and C-type natriuretic peptide (CNP) on the secretion of ACTH by perifused equine pituitary cells in an in vitro milieu intended to mimic the in vivo milieu in the horse. Corticotropin-releasing hormone (20 pM) and cortisol (0 or 100 nM) were perifused continuously and 7 pulses of arginine vasopressin (AVP; 10 nM) applied for 5 min at 30-min intervals. ANP (1 nM) or CNP (1 nM) were perifused continuously ...
Marsh AE, Barr BC, Madigan J, Lakritz J, Nordhausen R, Conrad PA.Neosporosis was diagnosed in an 11-year-old Quarter Horse gelding with clinical signs and diagnostic test results compatible with equine protozoal myeloencephalitis (EPM). Presumptive postmortem diagnosis of EPM attributable to Sarcocystis neurona infection is generally made on the basis of detecting an antibody titer to S neurona in the CSF or characteristic histologic lesions, even when parasites have not been specifically identified. Neosporosis was confirmed in the horse described here by use of immunohistochemical examination, in vitro culturing, and ultrastructural and molecular characte...
Gonzales DS, Jones JM, Pinyopummintr T, Carnevale EM, Ginther OJ, Shapiro SS, Bavister BD.The behaviour of bovine, equine and human blastocysts was studied in vitro by time-lapse videomicrography and computer imaging. This study revealed that cytoplasmic extensions of the trophectoderm ['trophectoderm projections' (TEP)] were expressed by embryos of all three species, prior to or during zona escape. Bovine and human blastocysts escaped their zonae with a combination of blastocoele expansion, collapse and re-expansion coupled with the penetration of the zona pellucida by TEP. In equine embryos, after several cycles of blastocoele expansion and collapse, trophectoderm ruptured the zo...
MacDonald MH, Benton HP.To determine the influence of activated equine neutrophils on sulfated glycosaminoglycan metabolism of equine articular cartilage in vitro. Methods: Articular cartilage explants harvested from the metacarpophalangeal joints of 7 horses. Methods: Proteoglycan degradation and synthesis were measured by release of glycosaminoglycan from the explants, and incorporation of [35S]sulfate into newly synthesized glycosaminoglycan. Results: Activated equine neutrophils significantly increased the release of glycosaminoglycan from explant matrix and the magnitude of that response was influenced by durati...
Sheoran AS, Holmes MA.Equine IgG possesses four well-defined subisotypes, designated IgGa, IgGb, IgGc and IgG(T) on the basis of their increasing anodal mobility in electrophoresis. However, the preparation of IgGa and IgGb reference proteins has not previously been reported. Certain bacterial cell wall proteins, termed protein A and protein G, have been used for purification of IgG subisotypes from the serum of domestic animals which, combined with other techniques utilising differences in the physico-chemical properties of the proteins, has allowed the purification of Ig isotypes. This paper describes purificatio...
Lauder RM, Huckerby TN, Nieduszynski IA.Fibromodulin has been isolated from bovine and equine articular cartilage and the attached keratan sulphate chains subjected to digestion by keratanase II. The oligosaccharides generated have been reduced and subsequently isolated by strong anion-exchange chromatography. Their structures have been determined by high-field 1H-NMR spectroscopy and high-pH anion-exchange chromatography. Both alpha(2-6)- and alpha(2-3)-linked N-acetylneuraminic acid have been found in the capping oligosaccharides, and, fucose which is alpha(1-3)-linked to N-acetylglucosamine has been found as a branch in both repe...
The Journal of physiologyDecember 1, 1996
Volume 497 ( Pt 2), Issue Pt 2 349-364 doi: 10.1113/jphysiol.1996.sp021773
Hanna WJ, Tsushima RG, Sah R, McCutcheon LJ, Marban E, Backx PH.1. The Na+ channel mutation associated with equine hyperkalaemic periodic paralysis (HPP) affects a highly conserved phenylalanine residue in an unexplored region of the alpha-subunit. This mutation was introduced into the rat skeletal muscle Na+ channel gene at the corresponding location (i.e. F1412L) for functional expression and characterization in Xenopus oocytes. 2. In comparison with wild-type (WT) channels, equine HPP channels showed clear evidence for disruption of inactivation: increased time-to-peak current, slowed rates of whole-cell current decay, significant increases in sustained...
The Journal of physiologyNovember 15, 1996
Volume 497 ( Pt 1), Issue Pt 1 19-29 doi: 10.1113/jphysiol.1996.sp021746
Ko WH, Chan HC, Wong PY.1. Anion secretion induced by capacitative Ca2+ entry through apical and basolateral membranes of cultured equine sweat gland epithelium was studied using the short-circuit current (Isc) technique. 2. Thapsigargin induced an increase in Isc that could be inhibited when external Ca2+ was chelated by EGTA. 3. The inhibition of the thapsigargin-induced Isc could be reversed by re-addition of Ca2+ to apical or basolateral solutions. The magnitude of the reactivated Isc depended predominantly on basolateral Ca2+ concentration. 4. The magnitude of the reactivated Isc upon basolateral Ca2+ addition i...
Crossett B, Allen WR, Stewart F.Large quantities of an unusual 19 kDa protein (p19) are secreted into the lumen of the uterus of the mare (Equus caballus) during the oestrous cycle and early pregnancy. p19 associates strongly with the acellular capsule that surrounds the young horse conceptus and is believed to be important in maintaining pregnancy. Here we report the complete cDNA sequence encoding p19, its expression patterns in horse tissues and a Southern blot analysis of the gene in horse DNA. The predicted amino acid sequence of the p19 cDNA demonstrated a signal peptide of 18 residues and a mature protein of 162 resid...
Madden CR, Shih DS.Sequential passage of the tissue culture-adapted prototype strain of EIAV in fetal donkey dermal (FDD) cell cultures generated a virus stock which exhibits cytopathic effects in FDD cell cultures. In this study, the effects of the long terminal repeat (LTR) region on virus replication and cytopathogenicity were examined. The FDD-adapted virus LTR was found to contain a number of base pair mutations and a large insertion within the U3 region in comparison with the previously characterized LTR, lambda12. Transient gene expression studies showed that basal promoter activity, in FDD cell cultures,...
Ball BA.There are considerable differences between mammalian species in the distribution and activity of ciliated cells within the oviduct, and limited information is available concerning either the distribution or activity of cilia within the equine oviduct. Patterns of ciliary activity were characterized in the ampulla and isthmus of oviducts recovered at 2 d after ovulation from 10 mares, and scanning electron microscopy was used to examine regional differences in the distribution of cilia in oviducts from 3 of these mares. Based upon the motility of 15 microm latex microspheres, ciliary activity w...
Marshall DE, Dumasia MC, Wooding P, Gower DB, Houghton E.Maternal endometrial and fetal allantochorionic tissues were separated manually from the placentae of seven healthy thoroughbred and three pony mares, ranging in gestational age from 100 to 318 days. The homogeneity of subcellular fractions prepared from these tissues was assessed initially using the marker enzymes, succinate dehydrogenase, NADPH cytochrome C reductase and lactate dehydrogenase for the mitochondrial, microsomal and cytosolic fractions, respectively. Light microscopy and histochemical analysis demonstrated that the separated fetal allantochorionic membrane, which is made up of ...
Dobrinski I, Ignotz GG, Thomas PG, Ball BA.To test the hypotheses that the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells (OEC) in vitro is mediated by glycoproteins, and that proteins with carbohydrate-binding properties are present in the periacrosomal plasma membrane of equine spermatozoa. Methods: 4 reproductively sound stallions, and 1 mare in estrus. Methods: In experiment 1a, fluorescent-labeled spermatozoa were cocultured with monolayers of OEC in the presence of 50 mM glucose, fructose, galactose, mannose, N-acetyl glucosamine, N-acetyl galactosamine, or N-acetyl neuraminic acid, or 10 mg of fet...
Caron JP, Tardif G, Martel-Pelletier J, DiBattista JA, Geng C, Pelletier JP.To determine whether matrix metalloprotease 13 (MMP-13; collagenase 3) is produced by equine chondrocytes and to investigate modulation of its expression by recombinant human interleukin 1 beta (rhIL-1 beta) and corticosteroids. Methods: Equine chondrocytes in monolayer culture were stimulated with rhIL-1 beta. Total RNA was extracted, purified, and reverse transcribed into DNA. Using appropriate primers, a putative MMP-13 fragment was amplified by polymerase chain reaction, and cloned into a bacterial vector. The resultant fragment was purified and sequenced, then was used to prepare a digoxi...
Sharma AK, Kathikeyan S, Kaur P, Singh TP, Yadav MP.Lactoferrin is an iron-binding glycoprotein with a molecular weight of 80 kDa. The protein has two iron binding sites. It has two structural lobes, each housing one Fe(3+) and the synergistic CO(3)(2-) ion. The protein was isolated from the colostrum/milk of mares maintained at National Research Centre on Equines, Hisar, India. The purified samples of the protein were crystallized using a microdialysis method. The protein was dialysed against low ionic strength buffer solution. Several crystal forms were obtained, out of which three were characterized which have cell dimensions as follows. For...
Landoni MF, Foot R, Frean S, Lees P.The objective of this study was to analyse the effects of 4 nonsteroidal anti-inflammatory drugs (NSAIDs) on the production of beta-glucuronidase (beta-glu), tumour necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), interleukin-1 (IL-1) and prostaglandin E2 (PGE2) by lipopolysaccharide (LPS)-stimulated equine synoviocytes. The agents studied were flunixin, tolfenamic acid, S(+)ketoprofen (KTP) and R(-)ketoprofen. LPS-induced release of beta-glu from synoviocytes was inhibited in a concentration dependent manner by all 4 compounds, tolfenamic acid being the most potent. Of the 2 KTP enant...
Cheng FP, Fazeli A, Voorhout WF, Marks A, Bevers MM, Colenbrander B.Peanut agglutinin (PNA) was used to assess the sperm acrosomal status and the acrosome reaction during gamete interaction in the equine species. PNA exclusively binds to the outer acrosomal membrane of stallion spermatozoa, as was established by transmission electron microscopy. Fluorescein isothiocyanate-PNA (FITC-PNA) labeling was used to monitor sperm acrosomal changes during a prolonged incubation period of 24 hours and during a 2-hours incubation in the presence of 5 microM calcium ionophore A23187. In addition, after a 4-hours preincubation in SP-TALP medium, sperm samples were incubated...
Hochi S, Maruyama K, Oguri N.The present study was designed to examine the suitability of ethylene glycol as a cryoprotectant for equine embryos. Blastocysts recovered nonsurgically from Day 6 donor mares were cryopreserved by conventional 2-step freezing in the presence of 10% ethylene glycol (EG), 10% glycerol (Gly), or 10% ethylene glycol + 0.1M sucrose (EG + Suc). After thawing, the EG and Gly were removed by a 6-step manner, and the EG + Suc was diluted to one fourth in the freezing straw. The postthaw blastocysts were transferred nonsurgically into the uteri of recipient mares on Days 4 to 7 after ovulation. Pregnan...
Research in microbiologyNovember 1, 1996
Volume 147, Issue 9 687-696 doi: 10.1016/s0923-2508(97)85116-9
Douet JP, Castroviejo M, Dodin A, Bébéar C.The haemolytic action of 125I-labelled thermostable direct haemolysin from Vibrio parahaemolyticus was studied on human and equine erythrocytes. In the first step, the haemolysin bound to the membranes of both erythrocyte species. This binding seemed temperature-independent. Then, for human erythrocytes, haemolysin produced cell disruption, and haemoglobin was released. Following this step, haemolysin was also released in a temperature-dependent manner. In contrast, equine erythrocytes were not disrupted, and no release of haemolysin occurred. The receptors of labelled haemolysin were analysed...
Kato H, Youn HY, Ohashi T, Watari T, Goitsuka R, Tsujimoto H, Hasegawa A.Using lipopolysaccharide (LPS)-stimulated equine peripheral blood mononuclear cell (PBMC) cDNA as a template, we performed polymerase chain reaction (PCR) amplification with equine interleukin-1 beta (IL-1 beta) specific primers. Electrophoresis of the PCR product on agarose gel revealed an additional smaller fragment that hybridized with an equine IL-1 beta cDNA probe. Sequencing of this fragment demonstrated that it was shorter than normal equine IL-1 beta cDNA by 162 nucleotides, which corresponded to exon 5 of the human and murine IL-1 beta genes. The deletion of 162 nucleotides did not re...
Ihler CF, Bjørn H.Ten stables were included in a study to evaluate two in vitro methods for the detection of anthelmintic resistance in cyathostomes by comparing a faecal egg count reduction test (FECRT) to a larval development assay (LDA) and an egg hatch assay (EHA). The LDA was used in seven stables and EHA in the last three. On the basis of FECR values, resistance to benzimidazoles was detected in eight of the ten small strongyle populations. Resistance to pyrantel pamoate and ivermectin was not detected. The mean concentrations that inhibited hatching in 50% of the eggs (EC50), using thiabendazole (TBZ) in...
Hoshino M, Kawata Y, Goto Y.GroEL interacts with proteins in denatured states and promotes their efficient folding. To understand the conformational features required for the substrate, we studied the interactions of GroEL with various derivatives of horse cytochrome c including porphyrin-cytochrome c, apo-cytochrome c, and the three fragments containing the heme group, i.e. fragments 1-65, 1-38 and 11-21. Size-exclusion chromatography was performed, taking advantage of the heme absorption of the fluorescence label. Under low-salt conditions, significant binding to GroEL was observed for porphyrin-cytochrome c, apo-cytoc...
Wilson SM, Rakhit S, Murdoch R, Pediani JD, Elder HY, Baines DL, Ko WH, Wong PY.Experiments were undertaken using cultured equine sweat gland epithelial cells that express purine receptors belonging to the P2U subclass which allow the selective agonist uridine triphosphate (UTP) to increase the concentration of intracellular free Ca2+ ([Ca2+]i). Experiments using pertussis toxin (Ptx), which inactivates certain guanine-nucleotide-binding proteins (G-proteins), showed that this response consisted of Ptx-sensitive and Ptx-resistant components, and immunochemical analyses of the G-protein alpha subunits present in the cells showed that both Ptx-sensitive (alpha i1-3) and Ptx...
Aurich JE, Kühne A, Hoppe H, Aurich C.Effects of seminal plasma on post-thaw motility and membrane integrity of cryopreserved horse spermatozoa were investigated. Carboxyfluorescein diacetate staining was used for the assessment of sperm membrane integrity. Adding 30% of seminal plasma from stallions with high post-thaw sperm motility to ejaculates from stallions with low post-thaw sperm motility increased progressive motility from 24.0 +/- 1.6 to 34.5 +/- 1.9% (P < 0.05) and membrane integrity from 27.0 +/- 2.1 to 34.3 +/- 2.3% membrane-intact spermatozoa (P < 0.05). Conversely, the addition of seminal plasma from stallions...
Sellon DC, Walker KM, Russell KE, Perry ST, Fuller FJ.Equine infectious anemia virus (EIAV) is a lentivirus that replicates predominantly in mature tissue macrophages. Viral expression is strongly influenced by the state of differentiation of the host cell. While blood monocytes can be infected, viral transcription is limited until the cell differentiates into a mature macrophage. Activation of mature macrophages infected with EIAV might also alter viral expression, presumably through binding of cellular transcription factors to viral nucleic acid sequences within the long terminal repeat (LTR). Using DNA amplification techniques, we compared LTR...
Gasser RB, Stevenson LA, Chilton NB, Nansen P, Bucknell DG, Beveridge I.Five species of equine strongyle belonging to the subfamily Strongylinae (Strongylus edentatus, S. equinus, S. vulgaris, Oesophagodontus robustus and Triodontophorus serratus) and 11 species belonging to the subfamily Cyathostominae (Poteriostomum imparidentatum, P. ratzii, Cylicocyclus insignis, Cc. leptostomus, Cc. nassatus, Cylicostephanus calicatus, Cs. longibursatus, Cs. goldi, Cyathostomum catinatum, Cy. labiatum and Cy. pateratum) were characterized using a polymerase chain reaction-linked restriction fragment length polymorphism technique (PCR-RFLP). Internal transcribed spacer ribosom...
Grøndahl C, Hyttel P.The nucleolus is believed to be the active site of rRNA synthesis in all eukaryotic cells. In preimplantation embryos, the embryonic genome is apparently more or less silent up to a species-specific developmental stage at which a major burst of transcription occurs. Here we report on nucleologenesis and some ultrastructural aspects of the onset of RNA synthesis in equine embryos during in vivo development. The zygotes and embryos up to blastocyst stages were surgically recovered from normally cycling mares. Mares were induced to ovulate by treatment with 3000 IU hCG and inseminated 20 and 34 h...
Almadhidi J, Moslemi S, Drosdowsky MA, Séralini GE.Aromatase (estrogen synthetase) is a steroidogenic enzyme complex which catalyzes the conversion of androgens to estrogens (termed aromatization). This enzyme was purified from adult equine testis to homogeneity by five chromatographic steps. The ability of purified and reconstituted equine aromatase to exhibit an estrogen 2-hydroxylase activity was tested and compared to testosterone aromatization. Enzymatic activities were assessed by tritiated water release from labelled estradiol and testosterone. Kinetic analysis of estradiol 2-hydroxylation showed an apparent K(m) of 23 microM and a V(ma...
May SA, Hooke RE, Lees P.Equine thymocytes, which respond to equine monocyte supernatants, do not respond to stimulation with recombinant human interleukin-1 alpha and beta, and equine synovial fibroblasts show a limited response in the form of prostaglandin E2 production without any evidence of neutral metalloproteinase production. Human interleukin-1 beta was about three to ten times as active on equine synovial cells as human interleukin-1 alpha in terms of prostaglandin E2 production. This preliminary evidence would suggest that there are qualitative and quantitative differences in the way recombinant human interl...
Sedrish SA, Venugopalan CS, Holmes EP, Koch CE, Moore RM.To determine in vitro vasomotor response of equine large colon arterial and venous rings with and without endothelium to vasodilator drugs, including dopamine (DOP), dopexamine (DPX), acepromazine (ACE), isoxsuprine (ISX), and nifedipine (NFP). Methods: 7 adult horses. Methods: Relaxation of large colon arteries and veins in response to vasodilating drugs was determined by measuring the change in tension of vessel rings when exposed to a cumulative concentration range (10(-8) to 10(-4)M) of each drug. Vessel rings, with and without endothelium, were mounted in organ baths, attached to a transd...
Adeyefa CA.The effects of the various growth factors with regard to the nutritional physiology of zoophilic dermatophytes isolated from horses are reported. The optimum temperature, pH and growth period for the fungal isolates were found to be 30 degrees-35 degrees C, 5-6 and 7-12 days respectively while the carbon and nitrogen sources utilised by the organisms were sucrose, fructose, maltose, L-arginine and calcium nitrate. The use of these factors in preparation of efficacious fungicides used in the treatment of ringworm infections in man and animals is discussed.
BMC research notesNovember 8, 2012
Volume 5 626 doi: 10.1186/1756-0500-5-626
Boudko SP, Ishikawa Y, Lerch TF, Nix J, Chapman MS, Bächinger HP.Hyperelastosis cutis is an inherited autosomal recessive connective tissue disorder. Affected horses are characterized by hyperextensible skin, scarring, and severe lesions along the back. The disorder is caused by a mutation in cyclophilin B. Results: The crystal structures of both wild-type and mutated (Gly6->Arg) horse cyclophilin B are presented. The mutation neither affects the overall fold of the enzyme nor impairs the catalytic site structure. Instead, it locally rearranges the flexible N-terminal end of the polypeptide chain and also makes it more rigid. Conclusions: Interactions of th...
McDonnell SM, Oristaglio Turner RM.Imipramine-induced ex copula ejaculates (11) and fractionated in copula ejaculates were collected from each of 5 pony stallions for freezing in 5-ml straws (6), using a modified Kenney glucose skim-milk extender (2). Initial post-thaw total and progressive motilities and daily post-thaw total and progressive motilities, as well as the number of days to reach 0 progressively motile spermatozoa, were also similar for the 2 methods of collection. The percentage of morphologically normal spermatozoa both before freezing and after thawing were also similar for in copula and ex copula ejaculates. Co...
Dean MF, Lee YW, Dastjerdi AM, Lees P.The basal rate of in vitro proteoglycan (PG) synthesis in explants of equine articular cartilage was subject to considerable variation in animals of the same age but was greater in younger than older animals. Synthesis of PGs in explant cultures was stimulated by a synthetic link peptide, identical in sequence to the N-terminus of the link protein (LP) of PG aggregates, in a similar manner to that demonstrated previously for human articular cartilage [Biochem. Soc. Trans. 25 (1997) 427; Arthritis Rheum. 41 (1998) 157]. Stimulation occurred in tissue from animals ranging from 1 to 30 years old ...
Rahnama S, Spence R, Vathsangam N, Baskerville CL, Bailey SR, de Laat MA, Anderson ST, Pollitt CC, Sillence MN.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...
Mancini RA, Ramanathan R.Injection-enhancement of beef with lactate improves color stability; however, the mechanism is unclear. Thus, our objectives were to assess the effects of sodium lactate on equine myoglobin redox stability in vitro. Oxymyoglobin at pH 5.6 (50mM sodium citrate) and pH 7.4 (50mM sodium phosphate) was incubated at 4°C with lactate (0, 5, 10, 100, or 200mM) and myoglobin redox form was determined using absorbance spectra. Metmyoglobin formation at pH 5.6 and 7.4 was significantly (P<0.05) decreased by lactate at concentrations of 100 and 200mM. In general, increasing lactate concentration from...
Todhunter RJ, Fubini SL, Wootton JA, Lust G.The effect of different doses of methylprednisolone acetate (MPA) on proteoglycan and collagen metabolism of articular cartilage from normal equine joints was tested in vitro. Methods: Cultured explants were treated with 0, 0.0004, 0.004, 0.04, 0.4 and 4.0 mg/ml (approximately 10(-6)-10(-2) M) MPA for 72 h. Proteoglycan synthesis was measured by incorporation of sodium [35S]sulfate into proteoglycans and proteoglycan degradation was measured by release of total and radiolabeled proteoglycan into the culture media. The size of the proteoglycans was assessed with size exclusion chromatography un...
Kirisawa R, Hosoi Y, Yamaya R, Taniyama H, Okamoto M, Tsunoda N, Hagiwara K, Iwai H.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...
Goudet G, Leclercq L, Bézard J, Duchamp G, Guillaume D, Palmer E.This study reports the follicular growth and oocyte competence for in vitro maturation and fertilization under the influence of circulating eCG. Three to 7 successive ultrasound-guided follicular punctures were performed on 4 pregnant mares from Day 23 until Day 75 of pregnancy and on 5 control mares whose embryonic vesicle was crushed on Day 22. All follicles larger than 5 mm were punctured 24 h after the largest follicle reached 18 mm. Expanded cumulus oocyte complexes (COCs) were stained at recovery to analyze the nuclear stage. Compact COCs were cultured in vitro for 46 h and either staine...
Gibb Z, Grupen CG, Maxwell WM, Morris LH.The fertility of sex-sorted, cryopreserved stallion sperm must be improved for the sex-sorting technology to be applied commercially. Objective: To optimise the conditions used to liquid store stallion sperm prior to sex-sorting and assess the fertility of sperm following sex-sorting and cryopreservation. Methods: Both in vitro experiment and randomised controlled trial in healthy, client-owned mares. Methods: Stallion ejaculates (n = 9) were diluted in either a skimmed milk (KMT) or BSA (I-BSA) based media to 25 × 106 sperm/ml directly (+SP25) or washed to remove seminal plasma and diluted t...
Ebert DW, Bertone AL, Roberts C.To determine whether irradiation with a low-intensity diode laser, which produces radiation at a wavelength of 810 nm, will induce nonthermal enhancement of chondrocyte metabolism. Methods: 144 grossly normal articular cartilage explants aseptically harvested from the femoral condyles of 6 adult horses. Methods: Treated cartilage explants were irradiated with a diode laser at 1 of 7 fluence levels that ranged from 8 to 1,600 J/cm2. Explants were incubated for 24 or 72 hours, labeled for 24 hours with [35S]Na2SO4, and assayed for newly synthesized sulfated glycosaminoglycan (GAG; measured incor...
McCarhey RN, Jeffcott LB, McCartney RN.Ten pairs of third metacarpal bones from Thoroughbred horses aged two to 12 years were used to estimate bone strength. Measurements of transverse cortical ultrasound velocity, cortical cross sectional area and bone mineral content were made using ultrasonic transmission velocity and single photon absorptiometry. These data were used to determine bone mineral density, compact bone density and modulus of elasticity. The results were compared with those measured by direct means or chemical analysis and satisfactory correlations were obtained between estimated and measured values. Single photon ab...
Mittelholzer C, Johansson I, Olsson AK, Ronéus M, Klingeborn B, Belák S.Recovery of infectious Equine arteritis virus (EAV) from the semen of persistently infected Swedish stallions was attempted by classical cell culture isolation and by transfection of extracted total RNA. Whereas virus from semen samples stored for several months at -20 degrees C or from extended semen could only be recovered by transfection of extracted RNA, isolation in cell culture was achieved readily with fresh, unextended semen stored at -70 degrees C or directly used after sampling. In parallel, the viruses were examined in the variable region of the large glycoprotein GP5 by nested RT-P...
Biedrzycki A, Markel MD, Brounts SH.To compare the in vitro biomechanical properties of a novel subcuticular stapling device to current methods of abdominal skin closure for equine abdominal surgery. Methods: In vitro randomized, matched design. Methods: Equine ventral median abdominal skin specimens (n = 24 horses). Methods: Subcuticular absorbable staples (SAS), metallic staples (MS), polyglyconate suture (PG), and nylon monofilament (NYL) were applied to longitudinally transected portions of equine ventral midline skin. Loads that resulted in an initial failure point and the ultimate failure load and mechanism were record...
Boschwitz JS, Timoney JF.A rapid method for purifying equine C3 which yields milligram quantities of pure C3 is described. Protein from equine plasma was selectively precipitated with polyethylene glycol, and the C3 was purified by anionic and cationic exchange HPLC. The yield from this procedure was 12%. The purified C3 was composed of an alpha chain (118 kD) and a beta chain (68 kD) linked by at least one disulfide bond, and it had an isoelectric point of 4.7. Amino acid analysis indicated a strong conservation of amino acid usage between equine and human C3. The N-terminal sequences of the alpha and beta chains wer...
Okólski A, Babusik P, Tischner M, Lietz W.Comparisons were made between 2 methods of oocyte recovery from the ovarian follicles of slaughtered mares: 500 oocytes (3 per mare) were obtained by aspiration of follicular fluid from ovaries of 162 mares, and 120 oocytes (8 per mare) by isolation and rupture of follicles from ovaries of 14 mares. In the oocytes recovered after rupture of follicles, 89.2% were morphologically unchanged, in comparison to 29.3% obtained by aspiration of follicular fluid. Stallion spermatozoa capacitated in vitro were tested on zona-free hamster oocytes. The stallion spermatozoa were washed in TCM-199 and prein...
Bousquet D, Guillomot M, Betteridge KJ.The capsule which surrounds the pre-attachment equine embryo has been compared with the zona pellucida (zp) that it replaces, as well as with the rabbit blastocyst coverings, by means of physicochemical and immunological methods. Trypsin solution at pH varying between 7.5 and 9.0 completely solubilized the capsule, as did Na borohydride. However, solutions of pH 2.0 or 12.0, urea, high temperature (65 degrees C, 60 min or 80 degrees C, 30 min), mercaptoethanol and dithiothreitol were able to solubilize the zp but not the capsule at the concentrations used. Indirect immunofluorescence on cryost...
Zerpa H, Vega F, Vasquez J, Ascanio E, Campos G, Sogbe E, Romero E, Ascanio M, García H.Endotoxaemia is a syndrome linked to the development of equine laminitis; however, the relationship between them is uncertain. The aim of this experiment was to evaluate the effect of an experimental acute sublethal endotoxaemia model on in vitro equine palmar digital vascular reactivity. Rings of arteries and veins of each forelimb were obtained from 11 clinically healthy horses submitted to two surgical procedures, 3 weeks apart. Before the second surgery, 0.25 microg/kg of lipopolysaccharide from Escherichia coli O55:B5 in saline, was administered i.v. in 30 min. After 3 h, the vessels were...
Ząbek T, Witarski W, Semik-Gurgul E, Szmatoła T, Kowalska K, Bugno-Poniewierska M.We investigated the activity of chondrogenic markers and variation of methylation patterns in equine cartilaginous cells cultivated in monolayer. The transcriptional and epigenetic effect of the long-term culture of chondrocytes has been evaluated using several passages of chondrocyte cell-lines derived from equine articular cartilage. Using 3 genes as endogenous control we tested the expression of 7 genes important for different stages of chondrocyte differentiation and maturation. CpG islands in RUNX3 locus were inspected for the evaluation of differential methylation state of passaged cell-...
Nourian AR, Mills PC, Pollitt CC.Microdialysis (MD) was used for continuous monitoring of the lamellar extracellular fluid (ECF) in six mature healthy Standardbred horses. MD probes were introduced into the lamellar tissue under local anaesthesia. Following intravenous injection of gentamicin (5mg/kg), MD and serum samples were collected for 24h and analysed using a sensitive ELISA test for gentamicin and fluorescence polarization immunoassay for urea concentrations. Calibration of probes was performed through in vivo urea recovery and in vitro gentamicin and urea recovery. Data obtained from different body compartments were ...
Lançoni R, Celeghini ECC, Gonella-Diaza AM, Júnior VG, de Carvalho CPT, Zoca GB, Garcia-Oliveros LN, Batissaco L, Oliveira LZ, de Arruda RP.This study aimed to assess the semen ubiquitin levels of stallions with good (GF) and poor semen freezability (PF) and to evaluate the relationship between sperm ubiquitination and sperm morphological defects. Five ejaculates from eight adult stallions (n = 40) were collected and cryopreserved. Then, the ubiquitin level in equine sperm cells was assessed by immunohistochemistry with epifluorescence microscopy, and sperm morphology was assessed by differential interference contrast microscopy. Sperm cells were classified according to the intensity (classification 1: from I to IV; I = very l...
Reardon R, Bailey R, Walmsley J, Heller J, Lischer C.The mechanical properties of equine cervical vertebrae joined by implants have not yet been reported. Locking compression plates (LCP) may provide a useful alternative system to the commonly used stainless steel kerf-cut cylinders (KCC) currently used for fixation of cervical vertebrae in horses. Objective: The objectives of this study were to establish a method for biomechanical testing of equine C4-C5 articulations and to compare the biomechanical properties of cadaveric spines stabilised with KCC and LCP. Methods: Twenty-four equine cadaveric cervical spines were size measured from radiogra...
Kareskoski AM, Palviainen M, Johannisson A, Katila T.For unknown reasons, stallion fertility and sperm longevity during cooled storage of semen vary markedly between individuals. Spermatozoa from individual stallions react differently to the presence, or the removal, of seminal plasma (SP). The aim was to evaluate differences in protein content in stallion seminal plasma with either a positive or a negative effect on sperm chromatin integrity during storage. Stallion semen samples from different ejaculate fractions were stored at 5°C for 24 hr. Sperm survival was assessed after storage using a sperm chromatin structure assay. Protein expressio...
Mok CH, MacLeod JN.Within developing synovial joints, interzone and anlagen cells progress through divergent chondrogenic pathways to generate stable articular cartilage and transient hypertrophic anlagen cartilage, respectively. Understanding the comparative cell biology between interzone and anlagen cells may provide novel insights into emergent cell-based therapies to support articular cartilage regeneration. The aim of this study was to assess the kinetics of gene expression profiles in these skeletal cell lines after inducing chondrogenesis in culture. Interzone and anlagen cells from seven equine fetuses w...
Blanch EW, Morozova-Roche LA, Hecht L, Noppe W, Barron LD.Vibrational Raman optical activity (ROA) spectra of the calcium-binding lysozyme from equine milk in native and nonnative states are measured and compared with those of the homologous proteins hen egg white lysozyme and bovine alpha-lactalbumin. The ROA spectrum of holo equine lysozyme at pH 4.6 and 22 degrees C closely resembles that of hen lysozyme in regions sensitive to backbone and side chain conformations, indicating similarity of the overall secondary and tertiary structures. However, the intensity of a strong positive ROA band at approximately 1340 cm(-1), which is assigned to a hydrat...
Hernández-Avilés C, Ramírez-Agámez L, Love CC, Friedrich M, Pearson M, Kelley DE, Beckham AMN, Teague SR, LaCaze KA, Brinsko SP, Varner DD.Under in vitro conditions, stallion sperm might preferentially use energy substrates that primarily undergo mitochondrial metabolism. The present study sought to determine the effects of glucose, pyruvate, lactate, or their combinations on the quality of stallion sperm subjected to cooled storage at different temperatures, when using a skim milk-based semen extender. In Experiment 1, no substrate (Control), glucose (40 mM; Glu-40), pyruvate (2 mM, 19.8 mM; Pyr-2, Pyr-19), lactate (2 mM, 19.8 mM; Lac-2, Lac-19, respectively), or their combinations (G/P/L-2 or G/P/L-19, respectively) were ...
Bentley AP, Barton MH, Lee MD, Norton NA, Moore JN.To determine which antimicrobials that are used to treat neonatal foals with septicemia attributable to Escherichia coli will minimize endotoxin release from bacteria and subsequent activity of inflammatory mediators while maintaining bactericidal efficacy. Methods: Blood samples from 10 healthy foals. Methods: Escherichia coli isolates A and B were isolated from 2 septicemic foals, and minimal inhibitory concentrations (MIC) were determined for 9 antimicrobials. Five of these antimicrobials were tested in vitro at 2 and 20 times their respective MIC. Whole blood or mononuclear cells grown in ...
Menozzi A, Pozzoli C, Poli E, Colla C, Placenza G, Bertini S.We investigated the effects of different selective α -adrenergic receptor (AR) agonists (detomidine, medetomidine, xylazine, and brimonidine) on the contractions of horse-isolated bronchi induced by electrical field stimulation (EFS) and by carbachol. No effects were observed on the contraction induced by carbachol, while α -AR agonists reduced EFS-evoked contractions in a concentration-related fashion. The rank order of potency (pD ) was brimonidine (7.40 ± 0.20) >medetomidine (7.09 ± 0.24) >detomidine (6.13 ± 0.55) >xylazine (4.59 ± 0.16). The maximal effects (E ) were -56.3% ...
