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.
Tharasanit T, Colleoni S, Lazzari G, Colenbrander B, Galli C, Stout TA.Oocyte cryopreservation is a potentially valuable way of preserving the female germ line. However, the developmental competence of cryopreserved oocytes is presently poor. This study investigated whether the morphology of the cumulus complex surrounding an immature equine oocyte and/or the oocyte's stage of maturation affect its cryopreservability. Compact (Cp) and expanded (Ex) cumulus oocyte complexes (COCs) were vitrified either shortly after recovery (germinal vesicle stage, GV) or after maturation in vitro (IVM); cryoprotectant-treated and -untreated non-frozen oocytes served as controls....
Blikslager AT, Yin C, Cochran AM, Wooten JG, Pettigrew A, Belknap JK.Recent reports indicate increased amounts of mRNA from inflammation-related genes in the prodromal stage of laminitis. Objective: Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) undergo distinct patterns of expression in equine laminae in the developmental stage (DEV) and acute clinical stage (LAM) of laminitis. Methods: Horses selected from an outbred population were placed into 1 of 4 groups: DEV (n = 5), CON-3h (control group for DEV, n = 5), LAM (n = 5) and CON-10h (control group for LAM, n = 5). Methods: Laminar and skin samples were obtained from (1) animals either undergoing leuko...
Boneker C, Kuiper H, Drögemüller C, Chowdhary BP, Distl O.The mammalian collagen, type IX, alpha 2 gene (COL9A2) encodes the alpha-2 chain of type IX collagen and is located on horse chromosome 2p16-->p14 harbouring a quantitative trait locus for osteochondrosis. We isolated a bacterial artificial chromosome (BAC) clone containing the equine COL9A2 gene and determined the complete genomic sequence of this gene. Cloning and characterization of equine COL9A2 revealed that the equine gene consists of 32 exons spanning approximately 15 kb. The COL9A2 transcript encodes a single protein of 688 amino acids. Thirty two single nucleotide polymorphisms (SNPs)...
Pagl R, Aurich C, Kankofer M.Activity of the anti-oxidative enzymes glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT), content of thiobarbituric acid reactive substances (TBARS) and SH-groups were determined in native stallion semen (n = 8 stallions). Semen was then diluted in Kenney extender, EquiPro((R)) extender either with or without addition of N-acetyl cysteine or phosphate-buffered saline (PBS) and stored for 72 h at 5 degrees C. Correlations between initial activity of enzymes and development of semen motility and membrane integrity were calculated. Activities of GSH-Px, SOD and CAT im...
Akcay E, Reilas T, Andersson M, Katila T.This study aimed to evaluate stallion sperm survival after 24 h of cooled storage in the presence of seminal plasma (SP) derived from the sperm-rich fractions (SRF) or sperm-poor fractions(SPF) of the ejaculate, without SP, or in the presence of SP from other stallions. Ejaculates were collected from four stallions using an automated phantom, which separated the semen into five cups. Centrifuged and washed spermatozoa from cup 2 (SRF) were mixed with skim milk extender to a concentration of 100 x 10(6) sperm/ml and then 1:1 (v/v) with SP from the stallion's own or another stallions' second (SP...
de Val N, Herschbach H, Potier N, Dorsselaer AV, Crichton RR.An essential difference between eukaryotic ferritins and bacterioferritins is that the latter contain naturally, in vivo haem as Fe-protoporphyrin IX. This haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two Met 52. However, in in vivo studies, a cofactor has been isolated in horse spleen apoferritin similar to protoporphyrin IX; in in vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haem. Studies of haemin (Fe(III)-PPIX) incorporation into horse spleen apoferritin have been carried out, which show that the me...
Mills PC, Cross SE.An increasing number of formulations are applied to equine skin, yet variable penetration can affect efficacy, or the incidence of adverse effects, or both. To investigate the effects of common methods of skin preparation on transdermal drug penetration in vitro, we clipped, harvested, and froze skin samples from 5 Thoroughbred geldings. Thawed samples were prepared as follows: control (no preparation); cleaned with aqueous chlorhexidine (Aq-C, 0.1% w/v); cleaned with alcoholic chlorhexidine (Al-C, 0.5% w/v); shaved (Sh); or tape-stripped (Ta) with the use of adhesive tape. The samples were th...
McD￮ LA, Anderson GI, Wright GM, Ryan DA.Bone cell cultures were evaluated to determine if osteogenic cell populations at different skeletal sites in the horse are heterogeneous. Osteogenic cells were isolated from cortical and cancellous bone in vitro by an explant culture method. Subcultured cells were induced to differentiate into bone-forming osteoblasts. The osteoblast phenotype was confirmed by immunohistochemical testing for osteocalcin and substantiated by positive staining of cells for alkaline phosphatase and the matrix materials collagen and glycosaminoglycans. Bone nodules were stained by the von Kossa method and counted....
Galli C, Colleoni S, Duchi R, Lagutina I, Lazzari G.Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo devel...
Jablonka-Shariff A, Roser JF, Bousfield GR, Wolfe MW, Sibley LE, Colgin M, Boime I.To study structure-activity relationships and the role of equine gonadotropins in the normal and pathophysiology of equine reproduction, the availability of purified hormones is essential. Previous expression studies in transfected CHO cells showed inefficient assembly of the human and bovine alpha and beta subunits, resulting in low levels of recombinant LH. The ability to express a single chain bearing genetically linked alpha and beta subunits bypasses this rate-limiting assembly step. A chimera was constructed by overlap PCR in which the carboxy terminal end of the eLHbeta subunit was gene...
Burnaugh L, Sabeur K, Ball BA.Low-level production of the superoxide anion (O2*-) is an important signal transduction event in sperm function including capacitation; however, excessive production of O2*- can be detrimental to sperm function. The objective of this study was to assess dihydroethidium (DHE) as a probe for O2*- in equine spermatozoa. Ejaculated spermatozoa were separated by centrifugation over a Percoll gradient (40:80), and loaded with DHE (2.0 microM) as well as with calcein-acetoxymethylester (CAM, 7.8 nM) to determine cell viability. In Experiment 1, cells were incubated with the xanthine-xanthine oxidase ...
Gandini M.To report a technique for closed-bowel 1-layer inverting end-to-end jejunojejunal anastomosis in horses. Methods: Experimental study. Methods: Fresh cadaveric jejunal segments from 12 horses. Methods: For each bowel segment a 1-layer closed and a 2-layer inverting end-to-end jejunojejunosotomy was created. Anastomosis construction time and anastomotic bursting pressure were measured and compared. Results: Closed-bowel anastomosis was significantly faster to create than a 2-layer technique. Luminal narrowing (<30%) was similar with both techniques and comparable with other inverting techniques....
Nieto JE, Dechant JE, Snyder JR.To evaluate single and double layer end-to-end anastomosis in equine jejunum. Methods: Experimental in vitro study. Methods: Mid-jejunal sections from 12 adult horses without gastrointestinal disease. Methods: Jejunal end-to-end anastomoses were performed by a continuous Lembert pattern or a simple continuous pattern oversewn with a Cushing pattern. Jejunal segments were distended with fluid at 1 L/min, and intraluminal pressure at failure, and mode of failure were recorded. Bursting pressure and bursting wall tension were calculated. Anastomosis construction time and degree of luminal reducti...
Vidal MA, Kilroy GE, Johnson JR, Lopez MJ, Moore RM, Gimble JM.To characterize equine bone marrow (BM)-derived mesenchymal stem cell (MSC) growth characteristics and frequency as well as their adipogenic and osteogenic differentiation potential. Methods: In vitro experimental study. Methods: Foals (n=3, age range, 17-51 days) and young horses (n=5, age range, 9 months to 5 years). Methods: Equine MSCs were harvested and isolated from sternal BM aspirates and grown up to passage 10 to determine cell-doubling (CD) characteristics. Limit dilution assays were performed on primary and passaged MSCs to determine the frequency of colony-forming units with a fibr...
Gandini M, Bertuglia A.To compare functional indices of end-to-end (EEA) jejunojejunal anastomosis using skin staples in horses with a 2-layer inverting hand-sewn technique. Methods: Experimental study. Methods: Jejunal segments from 8 fresh equine cadavers. Methods: For each bowel segment, 2 EEA anastomoses were created: one 2-layer hand-sewn and one 1-layer using skin staples. Time for anastomosis creation was recorded and compared. Lumen diameter of each anastomosis was measured on digital radiographs after intraluminal instillation of contrast medium and inflation of the jejunal segments to 14 mm Hg. Anastomotic...
Gygax D, Lischer C, Auer JA.To compare the precision of computer-assisted surgery with a conventional technique (CV) using a special guiding device for screw insertion into the distal sesamoid bone in horses. Methods: In vitro experimental study. Methods: Cadaveric forelimb specimens. Methods: Insertion of a 3.5 mm cortex screw in lag fashion along the longitudinal axis of intact (non-fractured) distal sesamoid bones was evaluated in 2 groups (8 limbs each): CV and computer-assisted surgery (CAS). For CV, the screw was inserted using a special guiding device and fluoroscopy, whereas for CAS, the screw was inserted using ...
Sod GA, Hubert JD, Martin GS, Gill MS.To compare biomechanical properties of a prototype 5.5 mm tapered shaft cortical screw (TSS) and 5.5 mm AO cortical screw for an equine third metacarpal dynamic compression plate (EM-DCP) fixation to repair osteotomized equine third metacarpal (MC3) bones. Methods: Paired in vitro biomechanical testing of cadaveric equine MC3 with a mid-diaphyseal osteotomy, stabilized by 1 of 2 methods for fracture fixation. Methods: Adult equine cadaveric MC3 bones (n=12 pairs). Methods: Twelve pairs of equine MC3 were divided into 3 groups (4 pairs each) for (1) 4-point bending single cycle to failure testi...
Saito S, Yokoyama K, Tamagawa T, Ishiwata I.We succeeded in the derivation and maintenance of pluripotent embryonic stem (ES) cells from equine and bovine blastocysts. These cells expressed markers that are characteristics of mouse ES cells, namely, alkaline phosphatase, stage-specific embryonic antigen 1, STAT 3 and Oct 4. We confirmed the pluripotential ability of these cells, which were able to undergo somatic differentiation in vitro to neural progenitors and to endothelial or hematopoietic lineages. We were able to use bovine ES cells as a source of nuclei for nuclear transfer and we generated cloned cattle with a higher frequency ...
Choi YH, Love LB, Varner DD, Hinrichs K.The effect of medium-to-embryo ratio on blastocyst development of equine embryos from oocytes with compact cumuli was evaluated in the present experiment. In addition, two methods for holding oocytes before in vitro maturation were compared. In Experiment 1, oocytes cultured with roscovitine for 16-18h before maturation were fertilized by intracytoplasmic sperm injection and cultured individually in 2.5, 5, 10 or 50microl droplets. In Experiment 2, oocytes were either cultured with roscovitine or held in a modified M199 with 20% serum at room temperature (EH treatment) for 16-18h, then matured...
Dacre KJ, McAleese SM, Knight P, McGorum BC, Pemberton AD.Mast cell mediators are believed to play a central role in inflammatory lung disorders such as human allergic and occupational asthma. Equine heaves is characterized by reversible neutrophilic airway inflammation and airway obstruction, primarily due to bronchospasm and mucus hypersecretion, following exposure of susceptible horses to organic stable dusts. As such, heaves shares many similarities with human occupational dust-induced asthma and therefore it is proposed that mast cells may also be implicated in the pathogenesis of heaves. Tryptase, a mast cell-specific proteinase, can be used as...
Betbeze CM, Wu CC, Krohne SG, Stiles J.To evaluate the in vitro antifungal properties of silver sulfadiazine (SSD) and natamycin against filamentous fungi isolated from eyes of horses with keratomycosis. Methods: Filamentous fungal isolates obtained from eyes of keratomycosis-affected horses. Methods: Fungal culture of ocular samples yielded 6 Fusarium spp; 7 Aspergillus spp; and 1 isolate each of Curvularia, Scopulariopsis, Penicillium, and Chrysosporium. For each fungal isolate, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of SSD and natamycin were determined. Results: For all 17 fungal isolat...
Ho EN, Kwok WH, Leung DK, Wan TS, Wong AS.Turinabol (4-chloro-17alpha-methyl-17beta-hydroxy-1,4-androstadien-3-one) is a synthetic oral anabolic androgenic steroid. As in the case of other anabolic steroids, it is a prohibited substance in equine sports. The metabolism of turinabol in human has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of turinabol in racehorses with an objective to identify the most appropriate target metabolites for detecting turinabol administration. For the in vitro studies, turinabol was incub...
López ML, Olea N, Retamal CA.Freeze-fracture replicas of stallion spermatozoa, collected from the proximal caput, corpus and cauda epididymides regions, were analyzed by electron microscopy to explore the distribution and density of intramembrane particles (IMP). Conspicuous differences in density and arrangement of the IMP were observed in the different topographical domains of mature and immature spermatozoa. A reduction of IMP, especially remarkable in the post-acrosomal domain, was observed in mature epididymal spermatozoa when compared with samples collected from ductuli efferentes. Some structural species-specific d...
Mills PC, Cross SE.The rate and regional differences for the penetration of fentanyl through equine skin was investigated in vitro using a commercial transdermal therapeutic system (TTS) or 'patch'. Skin collected from the thorax, groin and leg (dorsal metacarpal) regions of five horses was placed in diffusion cells and a fentanyl TTS applied to each skin sample. Drug penetration through each skin sample over 48 h measured using high performance liquid chromatography (HPLC). Cumulative penetration (microg/cm2) was plotted against time (h) and used to regress the steady state flux (microg/cm2/h) of fentanyl throu...
Nygaard TK, Liu M, McClure MJ, Lei B.Heme is a preferred iron source of bacterial pathogens. Streptococcus equi subspecies equi is a bacterial pathogen that causes strangles in horses. Whether S. equi has a heme acquisition transporter is unknown. Results: An S. equi genome database was blasted with the heme binding proteins Shp and HtsA of Streptococcus pyogenes, and found that S. equi has the homologue of Shp (designated SeShp) and HtsA (designated SeHtsA). Tag-free recombinant SeShp and SeHtsA and 6xHis-tagged SeHtsA (SeHtsAHis) were prepared and characterized. Purified holoSeShp and holoSeHtsA bind Fe(II)-protoporphyrin IX (h...
Staszyk C, Gasse H.Fibroblasts and cementoblasts in the periodontal ligament (PDL) of equine cheek teeth were harvested, and monocultures were obtained by means of a "selective detachment" procedure. Cells were characterized by morphological criteria and by immunostaining for vimentin, FVIII, pan-cytokeratin, smooth muscle actin, and pro-collagen. Cementogenic potential of the cells was determined by immunostaining for osteopontin and by histochemical detection of alkaline phosphatase. Equine periodontal fibroblasts (EPF) were spindle-shaped and polygonal. Equine dental cementoblasts (EDC) grew in cobblestone-li...
Dierks C, Mömke S, Drögemüller C, Leeb T, Chowdhary BP, Distl O.In this study, we present a comprehensive 5000-rad radiation hybrid map of a 40-cM region on equine chromosome 4 (ECA4) that contains quantitative trait loci for equine osteochondrosis. We mapped 29 gene-associated sequence tagged site markers using primers designed from equine expressed sequence tags or BAC clones in the ECA4q12-q22 region. Three blocks of conserved synteny, showing two chromosomal breakpoints, were identified in the segment of ECA4q12-q22. Markers from other segments of HSA7q mapped to ECA13p and ECA4p, and a region of HSA7p was homologous to ECA13p. Therefore, we have impro...
Li X, Zhou SG, Imreh MP, Ahrlund-Richter L, Allen WR.Inner cell mass (ICM) cells were isolated immunosurgically from day 7-8 horse blastocysts and, after proliferation in vitro for 15-28 passages, three lines of cells were confirmed to be embryonic stem (ES) cells by their continued expression of alkaline phosphatase activity and their ability to bind antisera specific for the recognized stem cell markers, SSEA-1, TRA-1-60, TRA-1-81, and the key embryonic gene Oct-4. When maintained under feeder cell-free conditions in vitro, the three lines of cells differentiated into cells of ectodermal, endodermal, and mesodermal lineages. However, they did ...
Argüelles D, Carmona JU, Pastor J, Iborra A, Viñals L, Martínez P, Bach E, Prades M.The aim of this study was to evaluate single and double centrifugation tube methods for concentrating equine platelets. Whole blood samples were collected from clinically normal horses and processed by use of single and double centrifugation tube methods to obtain four platelet concentrates (PCs): PC-A, PC-B, PC-C, and PC-D, which were analyzed using a flow cytometry hematology system for hemogram and additional platelet parameters (mean platelet volume, platelet distribution width, mean platelet component concentration, mean platelet component distribution width). Concentrations of transformi...
Bilińska B, Wiszniewska B, Kosiniak-Kamysz K, Kotula-Balak M, Gancarczyk M, Hejmej A, Sadowska J, Marchlewicz M, Kolasa A, Wenda-Rózewicka L.The purpose of this article was to summarize our results on the role of androgens and estrogens in human, rodent and equine testes and epididymides, in both, physiological and patological conditions, obtained in the space of the Solicited Project (084/PO6/2002) financially supported by the State Committee for Scientific Research during the last three years. Testosterone produced by Leydig cells of the testes is clearly the major androgen in the circulation of men and adult males of most mammalian species. However, androgen metabolites make up a significant fraction of total circulating steroid...
Vera L, Boyen F, de Visscher A, Vandenbroucke V, Vanantwerpen G, Govaere J.The use of commercial chromogenic agar plates for the rapid, easy and correct identification of equine endometritis-causing bacteria has been proposed. Preliminary tests in our lab revealed undescribed limitations. Therefore, we tested the ability of the Brilliance UTI agar, a commercially available chromogenic agar, to accurately identify bacteria causing equine endometritis. Objective: To 1) investigate whether bacteria present in the equine uterus are able to grow on this chromogenic agar plate, 2) determine whether these bacteria belong to the genera for which these agar plates were origin...
Conley AJ, Scholtz EL, Dujovne G, Cotterman RF, Legacki EL, Uliani RC, Alvarenga MA, Ball BA, Kalra B, Savjani GV, Kumar A.Studies in mares have examined serum inhibin concentrations using immuno-assays unable to distinguish dimeric inhibin-A from inhibin-B isoforms. Inhibin-A and inhibin-B immuno-assays were used to investigate concentrations in cyclic mares, young and old (6 vs 19 years old, respectively) mares following hemi-ovariectomy, mares during pregnancy and in mares with confirmed granulosa cell tumors (GCTs). Mares with inter-ovulatory intervals of 26 days had ovulatory peaks of inhibin-A averaging 80 pg/mL with a mid-cycle nadir of 5 pg/mL. Inhibin-A and inhibin-B concentrations were highly correlate...
Freeman DE.Accumulation of D-xylose by jejunal mucosa from healthy horses and rabbits was studied in vitro. When tissue sheets were incubated with 1 mM D-xylose for 60 minutes, mucosa from horses and rabbits accumulated D-xylose against a concentration gradient. There was no accumulation when equine specimens were incubated with 5 mM D-xylose. By comparison, equine jejunum accumulated D-glucose against a concentration gradient when incubated in 5 mM D-glucose. In equine and rabbit jejunum, 13.3 +/- 7.0% and 36 +/- 11.0%, respectively, of accumulated D-xylose was phosphorylated when sheets were incubated ...
Pitzer JB, Kaufman PE, Geden CJ, Hogsette JA.The ability of Spalangia cameroni Perkins, Spalangia endius Walker, and Muscidifurax raptorellus Kogan and Legner to locate and attack stable fly hosts was evaluated under laboratory conditions. Postfeeding third-instar stable fly larvae were released and allowed to pupate in two arena types: large 4.8 liter chambers containing a field-collected, soiled equine bedding substrate; or 120-ml plastic cups containing wood chips. At the time of fly pupariation, parasitoids were released and permitted 72 h to locate and attack hosts. On average, parasitism rates of freely accessible stable fly pupae ...
Verma RD, Venkateswaran KS, Sharma JK, Agarwal GS.Malleo-proteins from synthetic broth mallein of six strains of Pseudomonas mallei were separated by trichloroacetic acid precipitation, ammonium sulfate precipitation and Ultrogel AcA 34 gel filtration chromatography. When tested comparatively with Dutch PPD mallein as standard on P. mallei-sensitized and normal horses all the strains were found to be malleinogenic, trichloroacetic acid precipitated proteins were comparable to Dutch PPD mallein in potency and innocuity whereas ammonium sulfate-precipitated proteins elicited non-specific reactions. Ultrogel AcA 34 chromatographed high molecular...
Kilborne AH, Hussein H, Bertone AL.OBJECTIVE To investigate effects of hyaluronic acid (HA) or HA combined with chondroitin sulfate (CS) and N-acetyl-d-glucosamine (NAG) by use of a lipopolysaccharide (LPS) in vitro method. SAMPLE Monolayer cultures of synovial cells from 4 adult horses. PROCEDURES Synovial cell cultures were untreated or treated with HA alone or HA-CS-NAG for 24 hours, subsequently unchallenged or challenge-exposed with 2 LPS concentrations (20 and 50 ng/mL) for 2 hours, and retreated with HA or HA-CS-NAG for another 24 hours. Cellular morphology of cultures was evaluated at 0, 24 (before LPS), 26 (after LPS),...
Inkerman PA, Scott K, Runnegar MT, Hamilton SE, Bennett EA, Zerner B.Chicken, sheep, and horse liver carboxylesterases have been purified by procedures involving ammonium sulfate fractionation, ion-exchange chromatography and gel filtration on Sephadex. The actual yields of the procedures described were as follows: chicken, 1 g from 2 kg of liver powder (chloroform-acetone); sheep, 200 mg from 400 g of powder (chloroform-acetone); horse, 230 mg from 800 g of powder (acetone). The purified enzymes are free of non-carboxyl-esterase protein as shown by gel electrophoresis, although they do contain electrophoretic variants. The equivalent weight of the chicken enzy...
Sampaio B, Ortiz I, Resende H, Felix M, Varner D, Hinrichs K.Exposure to the calcium ionophore A23187 may present a "universal" sperm treatment for IVF, as it bypasses capacitation pathways. However, success in utilizing A23187 is variable, especially in equine spermatozoa. Notably, albumin is used during A23187 treatment but paradoxically is thought to suppress A23187 action. Essentially no critical data are available on the effects of A23187 and albumin concentrations, ratios, or addition protocols on changes in intracellular calcium ([Ca] ) in any cell type. To determine factors that affect the action of A23187 on [Ca] in equine and murine spermatozo...
Miragliotta V, Ipiña Z, Lefebvre-Lavoie J, Lussier JG, Theoret CL.Wound healing in horses is fraught with complications. Specifically, wounds on horse limbs often develop exuberant granulation tissue which behaves clinically like a benign tumor and resembles the human keloid in that the evolving scar is trapped in the proliferative phase of repair, leading to fibrosis. Clues gained from the study of over-scarring in horses should eventually lead to new insights into how to prevent unwanted scar formation in humans. cDNA fragments corresponding to CTNNB1 (coding for beta-catenin) and PECAM1, genes potentially contributing to the proliferative phase of repair,...
Stradaioli G, Sylla L, Zelli R, Verini Supplizi A, Chiodi P, Arduini A, Monaci M.The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas ...
Carter RA, Ericsson SA, Corn CD, Weyerts PR, Dart MG, Escue SG, Mesta J.The objective of this study was to determine if spermatozoal reduction of the dyes methylene green to colorless and resazurin to pink or colorless was associated with the fertility potential of an equine semen sample. Fifty samples from 38 stallions were evaluated for the number of spermatozoa per milliliter and number of motile sperm per milliliter. Methylene green (20 micrograms/mL of semen) or resazurin (85 micrograms/mL of semen) was added to 3-mL aliquots of semen. Semen samples were identified as having low fertility potential (< 200 x 10(6) total cells/mL and or = 200 x 10(6) total ...
Benamou AE, Marlin DJ, Callingham BC, Hiley RC, Lekeux R.There is currently little published information about the effects of endothelin-1 (ET-1), a potent endogenous spasmogen of vascular and airway smooth muscle, on pulmonary vasculature and airways or which ET receptor subtypes mediate ET-1-induced vasoconstrictive and bronchoconstrictive action in the horse. Objective: To investigate the effect of endothelin-1 (ET-1) on smooth muscle from isolated equine pulmonary artery and bronchus. In addition, the roles of ETA and ETB receptors in ET-1 mediated contraction in these tissues were assessed. Methods: The force generation of ring segments from pu...
Noé B, Poole AR, Mort JS, Richard H, Beauchamp G, Laverty S.Develop a species-specific ELISA for a neo-epitope generated by cathepsin K cleavage of equine type II collagen to: (1) measure cartilage type II collagen degradation by cathepsin K in vitro, (2) identify cytokines that upregulate cathepsin K expression and (3) compare cathepsin K with matrix metalloproteinase (MMP) collagenase activity in stimulated cartilage explants and freshly isolated normal and osteoarthritic (OA) articular cartilages. A new ELISA (C2K77) was developed and tested by measuring the activity of exogenous cathepsin K on equine articular cartilage explants. The ELISA was the...
Schramme MC, Boswell JC, Hamhougias K, Toulson K, Viitanen M.Numerous different techniques for injection of the navicular bursa have been described but there is little conformity between these descriptions. A literature review was performed and techniques for injection of the navicular bursa were categorised into 5 approaches. This study was performed to evaluate the reproducibility of each technique and to assess which technique was consistently most successful. Five inexperienced operators performed each technique on 5 cadaver forelimbs. Once a needle had been placed, a radiograph was taken to confirm placement was in accordance with the description o...
Odeh AI, Dascanio JJ, Caceci T, Bowen J, Eng LA.Phospholipids are an essential component of all mammalian cells; platelet activating factor (PAF=1-O-alkyl-acetyl-sn-glycero-3-phosphocholine) is a signalling phospholipid that has many biological properties in addition to platelet activation. PAF receptors have been detected on stallion spermatozoa; therefore, the aim of this study was to evaluate the effect of synthetic PAF on the motility, capacitation and the acrosome reaction of stallion spermatozoa. Treatment of ten stallion semen samples with 10(-4)-10(-13) mol PAF l(-1) resulted in significant differences in motility and capacitation (...
Olszewski MA, Robinson NE, Yu MF, Derksen FJ.During acute bouts of recurrent airway obstruction (heaves) in horses, neutrophils that are capable of increased production of reactive oxygen species accumulate in the airways. In the study reported here, the effect of hydrogen peroxide (H2O2; 1 microM to 0.1M), one of these reactive oxygen species products, on the responses of isolated trachealis muscle of horses was determined. Before and after incubation with H2O2, contractile responses to acetylcholine, electrical field stimulation (EFS), 127 mM KCl, and relaxation responses to isoproterenol and activation of the nonadrenergic noncholiner...
Choi YH, Velez IC, Macías-García B, Hinrichs K.In nuclear transfer (NT), exposure of donor cell chromatin to the ooplast cytoplasm may aid reprogramming; however, the length of exposure feasible is limited by the developmental life span of the oocyte. We examined the effect of duration of nucleus-cytoplasmic exposure before activation and of in vitro maturation (IVM) in equine NT. In experiment 1, 24 h IVM and a delay of 2, 5, or 8 h between reconstruction and activation yielded 4%, 15%, and 11% blastocysts, respectively. In experiment 2, a 5-h activation delay yielded 17% and 22% blastocysts with two donor cell lines. In experiment 3, usi...
Garvican ER, Vaughan-Thomas A, Redmond C, Clegg PD.Matrix degradation by metalloproteinases is considered a key feature in the loss of articular cartilage seen in many joint diseases. Membrane-type matrix metalloproteinase-3 (MT3-MMP) expression is elevated in human cartilage in end-stage osteoarthritis. We investigated whether MT3-MMP is similarly regulated in cartilage in two naturally occurring arthropathies in vivo and whether proinflammatory cytokines regulate its expression in vitro. MT3-MMP expression was evaluated in cartilage from horses with osteoarthritis and osteochondrosis and compared with age- and site-matched normal cartilage. ...
Pellegrini A, von Fellenberg R.The principal alpha-1-protease inhibitor of horse was fractionated by classical methods and analysed with a modified fibrinogen-agarose gel electrophoretic method of high sensitivity and resolving power. Starting with an electrophoretically homogeneous inhibitor in unfractionated serum, two isoinhibitor bands became apparent after fractionation with (NH4)2SO4 and DEAE-cellulose DE-52 ion-exchange chromatography. The isoinhibitors differed in electrophoretic migration and in the elution pattern from Sephadex G-100 gel filtration, but possessed identical antigenic determinants and enzyme specifi...
Stewart AS, Schaaf CR, Veerasammy B, Freund JM, Gonzalez LM.Equine intestinal epithelial stem cells (ISCs) serve as potential targets to treat horses with severe intestinal injury. The ability to isolate and store ISCs from intestinal biopsies creates an opportunity for both in vitro experiments to study ISC dynamics in a variety of intestinal diseases, and, in the future, utilize these cells as a possible therapy. If biopsies could be successfully stored prior to processing for ISCs, this would increase the availability of sample repositories for future experimental and therapeutic use. However, delayed culture of equine ISCs following prolonged sampl...
Cehak A, Wilkens MR, Guschlbauer M, Mrochen N, Schröder B, Feige K, Breves G.Transepithelial transport mechanisms play a key role in regulating the absorption and secretion of calcium (Ca(2+)) and inorganic phosphate (P(i)) in the gastrointestinal tract. Although intestinal disorders with imbalances in macromineral homeostasis are frequently observed in horses, available data on intestinal Ca(2+) and P(i) transport are limited. The aim of the present study was to characterize the intestinal Ca(2+) and P(i) transport functionally by using the in vitro radioisotope tracer technique with Ussing chambers and to identify components involved in Ca(2+) transport at both mRNA ...
Hormanski CE, Truax R, Pourciau SS, Folsom RW, Horohov DW.The in vitro stimulation of peripheral blood mononuclear cells (PBMC) with interleukin 2 (IL-2) results in the development of potent cytotoxic effector cells, referred to as lymphokine-activated killer (LAK) cells. LAK cells are capable of lysing a wide variety of autologous, allogeneic and xenogeneic tumor cells. The exact mechanism of target cell recognition by LAK cells remains unknown. LAK cell activity has been reported for a variety of domesticated species except the horse. We report here that IL-2-stimulated equine PBMC, which fail to lyse either human or murine tumor cell lines, exhibi...
Camp CJ, Leid RW.A method for the isolation of equine neutrophils was developed using metrizamide cushions. A purity of greater than 95% was routinely obtained with greater than 90% viability. These cells were radiolabeled and tested for their chemotactic response in Boyden chambers to zymosan-activated equine serum, the partially purified equine complement component C5a, and formyl-L-methionyl-L-leucyl-L-phenylalanine. The time and ionic requirements for chemotaxis of radiolabeled equine neutrophils were investigated and maximal movement was observed at 2 hours' incubation and 1.0 mM Ca and 0.5 mM Mg. Dinitro...
Trahan RA, Byron CR, Dahlgren LA, Pleasant RS, Werre SR.OBJECTIVE To compare the effects of 3 equimolar concentrations of methylprednisolone acetate (MPA), triamcinolone acetonide (TA), and isoflupredone acetate (IPA) on equine articular tissue cocultures in an inflammatory environment. SAMPLE Synovial and osteochondral explants from the femoropatellar joints of 6 equine cadavers (age, 2 to 11 years) without evidence of musculoskeletal disease. PROCEDURES From each cadaver, synovial and osteochondral explants were harvested from 1 femoropatellar joint to create cocultures. Cocultures were incubated for 96 hours with (positive control) or without (n...
Varner DD, Vaughan SD, Johnson L.Three ejaculates from each of 3 stallions were used to evaluate a computerized system (Hamilton-Thorn motility analyzer; HTMA) for measuring equine spermatozoal motility. Variance components (ejaculate-within-stallion, chamber-within-ejaculate, and microscopic field-within-chamber) were determined for each stallion after diluting ejaculates to 25 x 10(6) spermatozoa/ml with a skim milk-glucose seminal extender. The HTMA was compared with frame-by-frame playback videomicrography (VIDEO) for determining: percentage of spermatozoal motility and spermatozoal number in microscopic fields; curviline...
Onzere CK, Fry LM, Bishop RP, Da Silva M, Madsen-Bouterse SA, Bastos RG, Knowles DP, Suarez CE.Theileria equi is an obligate intracellular protozoan parasite that causes severe hemolytic anaemia in most equid species. Similar to other apicomplexan parasites, T. equi contains rhoptries whose contents have been implicated in host cell invasion and formation of the parasitophorous vacuole that is crucial for survival of the species within cells. Despite their importance, the composition of T. equi rhoptries and their role(s) in host cell invasion remain unexplored. To gain insight into these issues, we evaluated the expression, immunogenicity, and functional roles of two T. equi rhoptry-as...
Matyjaszek SA, Morton AJ, Freeman DE, Grosche A, Polyak MM, Kuck H.To examine the effects of flunixin meglumine (FM) on recovery of colonic mucosa from experimentally induced ischemia in horses. Methods: 14 research horses. Methods: Ischemia was induced in the colons of anesthetized horses for 2 hours. Afterward, horses received saline (0.9% NaCl) solution (12 mL, IV, q 12 h; n = 7) or FM (1.1 mg/kg, IV, q 12 h; 7) and were allowed to recover for 18 hours after termination of the ischemic event. Postoperative pain scores were recorded every 4 hours throughout the recovery period. At the end of the recovery period, horses were anesthetized, and ischemic and no...
Kinsley R, Pronost S, De Bock M, Temperton N, Daly JM, Paillot R, Scott S.Equine influenza is a major respiratory disease of horses that is largely controlled by vaccination in some equine populations. Virus-neutralising antibodies, the mainstay of the protective immune response, are problematic in assaying for equine influenza virus, as most strains do not replicate efficiently in cell culture. Surrogate measures of protective antibody responses include the haemagglutination inhibition (HI) test and single radial haemolysis (SRH) assay. For this study, a pseudotyped virus, bearing an envelope containing the haemagglutinin (HA) from the Florida clade 2 equine influe...
Beech J, Fletcher JE, Lizzo F, Johnston J.In vitro twitch characteristics of the semimembranosus muscle were evaluated in 9 clinically normal horses, in 15 horses with chronic intermittent rhabdomyolysis (CIR) and in 2 horses with myotonia. Effects of phenytoin on in vitro muscle twitch and clinical signs of CIR and myotonia were evaluated in these same horses. Times to 90% relaxation were prolonged in the horses with CIR (mean +/- SEM, 186 +/- 5.9 ms) and in 2 horses with myotonia (197 and 177 ms) compared with those in clinically normal horses (mean +/- SEM, 146 +/- 2.1 ms). Horses with CIR also had significantly (P less than 0.05) ...
Williams D, Siock P, Stenn K.A major challenge to the study of hair follicle growth is an appropriate assay system. Because equine mane follicles are large and noncurved, enabling easy dissection; are readily accessible from a single defined source; and possess a long anagen growth phase, we initiated a study of them in culture. As in our previous studies of human and sheep follicles (Dev Biol 165:469, 1994), we found in this system that transection level dictates the pattern of follicle growth in vitro: follicles transected below the sebaceous gland show a type 1 growth pattern (the shaft grows out with an adherent sheat...
