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.
Vasconcelos AB, Santana MA, Santos AM, Santoro MM, Lagares MA.Microscopy has been used in the routine evaluation of sperm metabolism. Nevertheless, it has limited capacity to preview male fertility. As calorimetry may be used to evaluate directly the metabolic activity of a biological system, the aim of this study was to use microcalorimetry as an additive method for sperm metabolism evaluation of cooled equine semen. Two ejaculates of four stallions were collected and motility, viability (eosin 3%) and membrane functional integrity (hyposmotic swelling test) of spermatozoa were evaluated. Sperm samples were processed following different protocols and th...
Morrell JM, Rodriguez-Martinez H, Johannisson A.This study compared the effect on semen quality of different handling methods used in the preparation of stallion semen doses for artificial insemination. The three methods were (i) extending the ejaculate to 30-50 x 10(6)/ml, (ii) single layer centrifugation (SLC) and (iii) sperm washing (centrifugation without a colloid). An additional treatment was to add seminal plasma (SP) in various proportions to some SLC preparations. The resulting samples were evaluated for sperm motility by computer assisted sperm analysis, membrane integrity using the Nucleocounter SP-100 and chromatin integrity by ...
Scarth JP, Clarke AD, Teale P, Pearce CM.Effective detection of the abuse of androgenic-anabolic steroids in human and animal sports often requires knowledge of the drug's metabolism in order to target appropriate urinary metabolites. 'Designer' steroids are problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of a toxicological profile. In this study, the in vitro metabolism of estra-4,9-diene-3,17-dione is reported for the first time. This is also the first study comparing the metabolism of a designer steroid in the three major species subject to sport's doping control; namely th...
Cheung WK, Working DM, Galuppo LD, Leach JK.Adipose stromal cells (ASC) are a promising alternative to progenitor cells from other tissue compartments because of their multipotential and capacity to retrieve significantly more progenitor cells. Initial cell samples are heterogeneous, containing a collection of cells that may contribute to tissue repair, but the sample becomes more homogeneous with each passage. Therefore, we hypothesized that the osteogenic potential of culture-expanded ASC would differ from uncultured ASC. Methods: Adipose tissue was collected from a yearling colt, and ASC were isolated and expanded using standard prot...
Groppelli E, Tuthill TJ, Rowlands DJ.Equine rhinitis A virus (ERAV) is genetically closely related to foot-and-mouth disease virus (FMDV), and both are now classified within the genus Aphthovirus of the family Picornaviridae. For disease security reasons, FMDV can be handled only in high-containment facilities, but these constraints do not apply to ERAV, making it an attractive alternative for the study of aphthovirus biology. Here, we show, using immunofluorescence, pharmacological agents, and dominant negative inhibitors, that ERAV entry occurs (as for FMDV) via clathrin-mediated endocytosis and acidification of early endosomes...
Sasaki N, Takakuwa J, Yamada H, Mori R.To evaluate effectiveness of allogeneic bone screws and pins for internal fixation of midbody transverse fractures of equine proximal sesamoid bones (PSBs) in vitro. Methods: 14 forelimbs from cadavers of 3-year-old Thoroughbreds. Methods: Allogeneic cortical bone fragments were collected from the limbs of a male Thoroughbred, and cortical bone screws were prepared from the tissue by use of a precision desktop microlathe programmed with the dimensions of a metal cortical bone screw. A midbody transverse osteotomy of each PSB was performed by use of a bone-shaping oscillating saw and repaired v...
Araujo JM, Araújo JV, Braga FR, Carvalho RO.Three isolates of predator fungi Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34), and Arthrobotrys robusta (I-31) were assessed in in vitro test regarding the capacity of prey infective larvae (L(3)) Strongyloides westeri. Compared to control, without fungus, there was a significant decrease (P 0.01) in vitro test. Linear regression coefficients of treated and control groups were -0.21 for control, -0.32 for D. flagrans, -0.34 for M. thaumasium, and -0.22 for A. robusta. In the following, isolates AC001 and NF34 were assessed in vivo regarding the capacity of supporting the pa...
Schmitz A, Thormann W, Moessner L, Theurillat R, Helmja K, Mevissen M.Ketamine, an injectable anesthetic and analgesic consisting of a racemic mixture of S-and R-ketamine, is routinely used in veterinary and human medicine. Nevertheless, metabolism and pharmacokinetics of ketamine have not been characterized sufficiently in most animal species. An enantioselective CE assay for ketamine and its metabolites in microsomal preparations is described. Racemic ketamine was incubated with pooled microsomes from humans, horses and dogs over a 3 h time interval with frequent sample collection. CE data revealed that ketamine is metabolized enantioselectively to norketamine...
Clulow JR, Evans G, Maxwell WM, Morris LH.The aim of the present study was to evaluate the potential oocyte binding ability and functional integrity of fresh or frozen-thawed, sex-sorted or non-sorted stallion spermatozoa. In the absence of effective IVF procedures in the horse, a heterologous sperm-binding assay was used as an indicator of fertilising capacity to assess differences in the ability of stallion spermatozoa to bind to bovine oocytes. The functional integrity of four treatment groups was assessed: (1) fresh non-sorted spermatozoa; (2) fresh sex-sorted spermatozoa; (3) frozen-thawed non-sorted spermatozoa; and (4) frozen-t...
Zerpa H, Berhane Y, Woodcock H, Elliott J, Bailey SR.A decrease in environmental temperature can directly affect the contractility of cutaneous vasculature, mediated in part by alpha(2)-adrenoceptors. Most of the cellular mechanisms underlying the cooling-enhanced contractility to alpha(2)-adrenoceptor agonists have been reported in cutaneous arteries but little information is available on cutaneous veins. To investigate the cellular mechanisms associated with the cooling-enhanced contraction to UK-14304 (alpha(2)-adrenoceptor agonist), isolated equine digital veins (EDVs) were studied at 30 degrees C and 22 degrees C. The effects of inhibitors ...
Bellone RR, Forsyth G, Leeb T, Archer S, Sigurdsson S, Imsland F, Mauceli E, Engensteiner M, Bailey E, Sandmeyer L, Grahn B, Lindblad-Toh K, Wade CM.Leopard Complex spotting occurs in several breeds of horses and is caused by an incompletely dominant allele (LP). Homozygosity for LP is also associated with congenital stationary night blindness (CSNB) in Appaloosa horses. Previously, LP was mapped to a 6 cm region on ECA1 containing the candidate gene TRPM1 (Transient Receptor Potential Cation Channel, Subfamily M, Member 1) and decreased expression of this gene, measured by qRT-PCR, was identified as the likely cause of both spotting and ocular phenotypes. This study describes investigations for a mutation causing or associated with the Le...
Turowska A, Pajak B, Godlewski MM, Dzieciatkowski T, Chmielewska A, Tucholska A, Banbura M.Viruses can reorganize the cytoskeleton and restructure the host cell transport machinery. During infection viruses use different cellular cues and signals to enlist the cytoskeleton for their mission. However, each virus specifically affects the cytoskeleton structure. Thus, the aim of our study was to investigate the cytoskeletal changes in homologous equine dermal (ED) and heterologous Vero cell lines infected with either equine herpesvirus 1 (EHV-1) strain Rac-H or Jan-E. We found that Rac-H strain disrupted actin fibers and reduced F-actin level in ED cells, whereas the virus did not infl...
Mienaltowski MJ, Huang L, Bathke AC, Stromberg AJ, MacLeod JN.Human and equine cell transplant strategies for cartilage lesions usually result in scar tissue that is similar to what is produced naturally during the repair process. In this study, culture-expanded de-differentiated chondrocytes and primary bone marrow stromal cells at a pre-transplantation time-point were compared along with neonatal cartilage to repair tissue. Transcriptional profiling using a 9413-probeset equine-specific cDNA microarray and targeted real-time quantitative polymerase chain reaction validation were used to characterize relationships between these cell types and repair tis...
Lagares MA, Castanheira PN, Amaral DC, Vasconcelos AB, Veado JC, Arantes RM, Stahlberg R.The aim of the present study was to evaluate the in vitro viability of equine embryos vitrified in three different solutions. Day 6 and 6.5 embryos were measured and morphologically evaluated. Only grade 1 or 2 morulae and early blastocysts were vitrified. Eighteen embryos were distributed in Group 1: 40 percent ethylene glycol in PBS, Group: 2 and 3: 40 percent ethylene glycol, 18 percent Ficoll, 0.3M sucrose or 0.3M trehalose in PBS, respectively. The vitrified embryos were loaded individually into 0.25 ml straws, which were cooled and immersed in liquid nitrogen. After warming at 20 degree ...
Nielsen SB, Wilhelm K, Vad B, Schleucher J, Morozova-Roche LA, Otzen D.The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to alpha-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of alpha-lactalbumins. Recently, EL multimeric complexes with oleic acids (ELOAs) were shown to possess tinctorial and morphological properties, similar to amyloidal aggregates, and to be cytotoxic. ELOA's interactions with phospholipid membranes appear to be central to its biological action, similar to human alpha-lactalbumin made lethal to tumor cells. ...
Brooks AC, Menzies-Gow N, Bailey SR, Cunningham FM, Elliott J.Hypoxia may enhance the deleterious effects of lipopolysaccharide (LPS) in the endotoxaemic horse. This study has examined some of the actions of LPS and hypoxia, alone and in combination, on cultured equine digital vein endothelial cells (EDVEC) and the signalling molecules involved. Methods: EDVEC were exposed to LPS, 5% O(2) and LPS then 5% O(2) for up to 24 h. HIF-1alpha stabilisation, neutrophil adhesion and EDVEC permeability were assessed by immunoblotting, measurement of myeloperoxidase and movement of FITC-dextran, respectively. Pharmacological inhibitors were used to assess the roles...
Visser MB, Pollitt CC.Most research to date involving laminins and extracellular matrix protein function in both normal and pathological conditions involves in vitro culture of keratinocytes. Few methods are established to allow for prolonged propagation of keratinocytes from equine tissues, including the hoof lamellae. In this study we modified cell isolation and culture techniques to allow for proliferation and sub-culturing of equine lamellar keratinocytes. Additionally, the production and processing of extracellular matrix molecules by skin and lamellar keratinocytes were studied. Results: Physical and proteoly...
Meehan M, Burke FM, Macken S, Owen P.Streptococcus equi possesses a haem-uptake system homologous to that of Streptococcus pyogenes and Streptococcus zooepidemicus. The system consists of two ligand-binding proteins (Shr and Shp) and proteins (HtsA-C) with homology to an ABC transporter. The haem-uptake system of S. equi differs from that of S. pyogenes and S. zooepidemicus in that Shr is truncated by two-thirds. This study focused on the SeShr, SeShp and SeHtsA proteins of S. equi. Analysis of shr, shp and shphtsA knockout mutants showed that all three proteins were expressed in vitro and that expression was upregulated under co...
Lewis AJ, Sod GA, Burba DJ, Mitchell CF.To compare compression pressure (CP) of 6.5 mm Acutrak Plus (AP) and 4.5 mm AO cortical screws (AO) when inserted in simulated lateral condylar fractures of equine 3rd metacarpal (MC3) bones. Methods: Paired in vitro biomechanical testing. Methods: Cadaveric equine MC3 bones (n=12 pair). Methods: Complete lateral condylar osteotomies were created parallel to the midsagittal ridge at 20, 12, and 8 mm axial to the epicondylar fossa on different specimens grouped accordingly. Interfragmentary compression was measured using a pressure sensor placed in the fracture plane before screw placement for ...
Sod GA, Riggs LM, Mitchell CF, Hubert JD, Martin GS.To compare in vitro monotonic biomechanical properties of an axial 3-hole, 4.5 mm narrow dynamic compression plate (DCP) using 5.5 mm cortical screws in conjunction with 2 abaxial transarticular 5.5 mm cortical screws inserted in lag fashion (DCP-TLS) with 3 parallel transarticular 5.5 mm cortical screws inserted in lag fashion (3-TLS) for the equine proximal interphalangeal (PIP) joint arthrodesis. Methods: Paired in vitro biomechanical testing of 2 methods of stabilizing cadaveric adult equine forelimb PIP joints. Methods: Cadaveric adult equine forelimbs (n=15 pairs). Methods: For each fore...
Tong T, Bai Y, Liu G, Wang Q, Zhang W, Xiao Y, Xu S, Liu N, Yang T, Wu D.IL-18 is a cytokine originally discovered as an important modulator of immune responses and subsequently shown to be pleiotropic. In this report, we expressed the recombinant equine mature interleukin-18 (rEMIL-18) in E. coli and purified it by nickel affinity gel column chromatography. Purified rEMIL-18 had biological activity commensurate with recombinant human IL-18, as determined by its synergistic effect with recombinant human IL-12 (rhIL-12) on the induction of IFN-gamma gene expression in equine peripheral blood mononuclear cells (PBMC). Following intraperitoneal (i.p.) immunization of ...
Hosaka YZ, Uratsuji T, Ueda H, Uehara M, Takehana K.This aim of this study was to determine the characteristic differences in tendinocytes derived from three sites of the equine superficial digital flexor tendon (SDFT)-proximally the myotendinous junction (MTJ), mid-metacarpal (mM) and osteotendinous junction (OTJ)-in morphology, proliferation, and ability for synthesis of collagen and matrix metalloproteinases (MMPs). Little difference was observed in cell proliferation. Addition of tumor necrosis factor (TNF) alpha to the culture medium resulted in increased collagen synthesis by tendinocytes from all three sites. The amount of collagen synth...
Jacobson CC, Choi YH, Hayden SS, Hinrichs K.Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to ...
Zhang J, Stein DA, Timoney PJ, Balasuriya UB.A significant consequence of equine arteritis virus (EAV) infection of horses is persistence of the virus in a variable percentage of infected stallions. We recently established an in vitro model of EAV persistence in cell culture for the purpose of furthering our understanding of EAV biology in general and viral persistence in the stallion in particular. In this study we investigated whether persistently infected HeLa cells could be cured of EAV infection by treatment with an antisense peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) designed to target the 5'-terminal region o...
Ma G, Lu C, Osterrieder N.A single amino acid variation in the equine herpesvirus type 1 (EHV-1) DNA polymerase (Pol) (D752/N752) determines its neuropathogenic potential. Here, an EHV-1 strain RacL11 mutant with a deletion of Pol residue 752 was constructed. The deletion virus was then repaired to encode D752 or N752, respectively. The Delta752 mutant virus replicated with kinetics indistinguishable from those of D752 and N752 viruses. In addition, we could demonstrate that the deletion mutant was significantly more resistant to aphidicolin, a drug targeting Pol, compared with the N752 but not the D752 variant. In equ...
Hodson DJ, Townsend J, Gregory SJ, Walters C, Tortonese DJ.A combined suppressive effect of prolactin (PRL) and dopamine on the secretion of luteinising hormone (LH) at the level of the pituitary gland has been identified in sheep, a short-day breeder. However, little is known about the role of PRL in the intra-pituitary regulation of the gonadotrophic axis in long-day breeders. In the present study, we investigated the effects of PRL on LH and follicle-stimulating hormone (FSH) secretion during the equine annual reproductive cycle. Horse pituitaries were obtained during the breeding season (BS) and nonbreeding season (NBS). Cells were dispersed, plat...
Jarrett M, Hibbert DB, Osborne R, Young EB.The analysis of total carbon dioxide (TCO(2)) in equine plasma is conventionally done in Australia and elsewhere using Beckman Synchron EL-ISE(R) analysers. This instrument is no longer being manufactured and has not been supported by the supplier since December 2008. For testing for TCO(2) to continue, it is necessary to evaluate and commission alternative instrumentation. In this paper, we compare the Beckman Synchron EL-ISE, the Beckman Synchron CX5, the Beckman UniCel DxC 600 and the Randox Daytona instruments. Results indicate that all four instruments perform in accordance with the manuf...
Desantis S, Ventriglia G, Zizza S, Nicassio M, Valentini L, Di Summa A, Lacalandra GM.Stallion sperm from semen collected in Southern Italy during the breeding (June-July) and non-breeding (December-January) periods were analyzed by means of twelve lectins to evaluate the glycoconjugate pattern and to verify whether there are any seasonal differences in the glycosylation pattern of the sperm glycocalyx. The acrosomal cap showed reactivity for Maackia amurensis (MAL II), Sambucus nigra (SNA), Arachis hypogaea (PNA), Glycine max (SBA), Helix pomatia (HPA), Canavalia ensiformis (Con A) Triticum vulgaris (WGA), and Griffonia simplicifolia isolectin II (GSA II) in breeding and non-b...
Mobasheri A, Lewis R, Maxwell JE, Hill C, Womack M, Barrett-Jolley R.Chondrocytes possess the capacity to transduce load-induced mechanical stimuli into electrochemical signals. The aim of this study was to functionally characterize an ion channel activated in response to membrane stretch in isolated primary equine chondrocytes. We used patch-clamp electrophysiology to functionally characterize this channel and immunohistochemistry to examine its distribution in articular cartilage. In cell-attached patch experiments, the application of negative pressures to the patch pipette (in the range of 20-200 mmHg) activated ion channel currents in six of seven patches. ...
Nemoto M, Imagawa H, Tsujimura K, Yamanaka T, Kondo T, Matsumura T.Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was applied to detection of equine rotavirus. Because equine rotavirus of the single P genotype, P[12], is predominant in the equine population worldwide, an RT-LAMP primer set was designed to target the genotype P[12] sequence and thus detect equine rotavirus. The detection limit of the RT-LAMP assay was 10(3) copies of viral RNA, whereas that of semi-nested RT-PCR for genotype P[12] was 10(5) copies. The RT-LAMP assay specifically amplified genotype P[12] but did not amplify the other P genotype strains. The RT-LAMP assay...
Gilger BC, Yang P, Salmon JH, Jaffe GJ, Allen JB.To determine whether a chemokine (RANTES)-like protein expressed by ciliary epithelium plays a role in uveitis. Methods: 3 clinically normal horses intradermal, 5 eyes from 5 horses with recurrent uveitis, and 10 normal eyes from 5 age- and sex-matched horses. Methods: Cross-reactivity and sensitivity of recombinant human (rh)-regulated upon activation, normal T-cell expressed and secreted (RANTES) protein were evaluated in horses by use of intradermal hypersensitivity reactions and a chemotaxis assay. Aqueous humor and ciliary body of eyes from clinically normal horses and horses with uveitis...
Textor JA, Murphy KC, Leach JK, Tablin F.To compare fiber diameter, pore area, compressive stiffness, gelation properties, and selected growth factor content of platelet-rich fibrin gels (PRFGs) and conventional fibrin gels (FGs). Methods: PRFGs and conventional FGs prepared from the blood of 10 healthy horses. Methods: Autologous fibrinogen was used to form conventional FGs. The PRFGs were formed from autologous platelet-rich plasma of various platelet concentrations (100 × 10³ platelets/μL, 250 × 10³ platelets/μL, 500 × 10³ platelets/μL, and 1,000 × 10³ platelets/μL). All gels contained an identical fibrinogen concentra...
Rossi M, Gonzalez-Castro R, Falomo ME.Different additives have been tested in cooled stallion sperm, in order to maintain sperm quality and to ameliorate the decrease in sperm fertility potential. In several species, caffeine and pentoxifylline promote sperm motility by increasing energy production. We evaluate the effects of caffeine and pentoxifylline when added to stallion sperm before or after cooling. Three ejaculates from five stallions each were processed and resuspended in skim milk extender. Caffeine (5 mM), pentoxifylline (3.5 mM), or both additives combined were included to sperm before or after cooling (4°C for 24 ...
Ménard M, Nauc V, Lazure C, Vaillancourt D, Manjunath P.A family of bull seminal plasma (BSP) phospholipid-binding proteins (BSP proteins), potentiate heparin- and HDL-induced capacitation. The homologous proteins have been purified from stallion and boar seminal plasma, and detected in low concentrations in other mammalian seminal plasma. In this study, we developed a new isolation method for mammalian seminal plasma choline phospholipid-binding proteins wherein they are present in low concentrations. The method is based on the interaction of this family of proteins with egg yolk low-density lipoprotein fraction (LDF). In order to demonstrate the ...
Bochröder B, Schubert R, Bödeker D.Discs of stripped mucosa from the proximal ventral colon were prepared immediately after slaughter of 8 Shetland cross-breed ponies. The mucosae were fixed in incubation chambers and used in incubation experiments to study the transmucosal fluxes of the amino acids lysine, histidine and arginine (150 min) and of ammonia (90 min). When the amino acid concentrations in the mucosal solution were in the physiological range (2.8-3.0 mmol/l) no transport to the serosal side of the tissue was found. When the concentrations were raised 10-fold, less than 2% of the mucosal amino acid pool was recovered...
Pedreros M, Ratto M, Guerra M.In the present study, we analysed the molecular mechanism(s) by which melatonin directly affects ovarian function in the mare. In Experiment 1, follicles and corpora lutea (CL) were collected from slaughterhouse ovaries and analysed for melatonin (MT(1)) receptor mRNA and protein. In Experiment 2, CL were collected from slaughterhouse ovaries and cultured in Dulbecco's modified Eagle's medium-F12 medium (control medium) supplemented with 50 ng mL(-1) equine chorionic gonadotrophin (eCG), 1 nM-1 μM melatonin, 1 μM forskolin or 1 μM luzindole. Explants were cultured for 3 h in the presence of...
Catalán J, Yánez-Ortiz I, Gacem S, Papas M, Bonet S, Rodríguez-Gil JE, Yeste M, Miró J.Previous research has determined that irradiation of mammalian sperm with red light increases motility, mitochondrial activity, and fertilization capacity. In spite of this, no study has considered the potential influence of the color of the straw and the extender used. Therefore, this study tests the hypothesis that the response of mammalian sperm to red light is influenced by the color of the straw and the turbidity/composition of the extender. Using the horse as a model, 13 ejaculates from 13 stallions were split into two equal fractions, diluted with Kenney or Equiplus extender, and stored...
Lewis N, Schnauffer K, Hinrichs K, Morganti M, Troup S, Argo C.The use of time-lapse imaging (TLI) in the evaluation of morphokinetics associated with invitro developmental competence is well described for human, cattle and pig embryos. It is generally accepted that embryos that complete early cleavage sooner are more likely to form blastocysts and that timing of later events, such as blastocyst formation and expansion, are predictive of implantation potential and euploid status. In the horse, morphokinetics as a predictor of developmental competence has received little attention. In this study we evaluated the morphokinetics of early equine embryo develo...
Wahl K, Adams SB, Moore GE.To assess fetlock joint contamination with tissue debris and hair after arthrocentesis. Methods: Experimental. Methods: Fetlock joint tissues (n = 10 horses). Methods: Soft tissue flaps including the joint capsule were dissected from the dorsal fetlock joints of 7 anesthetized horses leaving an intact proximal base. Needles inserted through the tissue flaps were flushed into tissue cell culture plates and examined for debris. Studies were repeated on excised fetlock tissue preparations after being stored for 5 days. Variables included gauge and type of needle, insertion of spinal needles with ...
Beaumont RE, Smith EJ, Zhou L, Marr N, Thorpe CT, Guest DJ.We investigated how Interleukin 1 beta (IL-1β) impacts equine tenocyte function and global gene expression in vitro and determined if these effects could be rescued by pharmacologically inhibiting nuclear factor-κB (NF-B) or interleukin 1 signalling. Equine superficial digital flexor tenocytes were cultured in three-dimensional (3D) collagen gels and stimulated with IL-1β for two-weeks, with gel contraction and interleukin 6 (IL6) measured throughout and transcriptomic analysis performed at day 14. The impact of three NF-B inhibitors on gel contraction and IL6 secretion were measured in 3D ...
McGlinchey L, Boone LH, Munsterman AS, Coleridge MOD, Rosanowski SM, Farag R, Hanson RR.To evaluate the effect of exposure to a balanced electrolyte solution (BES), or equine abdominal fat on the knot-holding capacity (KHC), relative knot security (RKS), weight, and volume of forwarder knots versus surgeon's knots. Methods: 315 knots tied and tested in vitro. Methods: United States Pharmacopeia size-3 polyglactin 910 suture exposed to air (dry [control]), equine abdominal fat (fat-exposed), or BES (BES-exposed) was used to tie forwarder knots with 2, 3, and 4 throws and surgeon's knots with 5, 6, 7, and 8 throws. A universal materials testing machine was used to test the tensile ...
Grant DM, Macedo A, Toms D, Klein C.Preimplantation equine embryos synthesize and secrete fibrinogen, which is a peculiar finding as fibrinogen synthesis almost exclusively occurs in the liver. This study investigated the hypothesis that conceptus-derived fibrinogen mediates cell adhesion during fixation. On day 21 of pregnancy, five integrin subunits, including ITGA5, ITGB1, ITGAV, and ITGB1, displayed significantly higher transcript abundance than on day 16 of pregnancy. Endometrial epithelial cells adhered to fibrinogen in an integrin-dependent manner in an in vitro cell adhesion assay. Bilaminar trophoblast and allantochorio...
Thomas PG, Ignotz GG, Ball BA, Miller PG, Brinsko SP, Currie B.Oviduct epithelial cells (OEC) increasingly are used to support embryonic development and to study gamete interactions with the female reproductive tract in vitro. This series of experiments was designed to characterize monolayers derived from oviduct epithelium. Epithelial cells harvested from the isthmus and ampulla of the oviducts of five estrous mares were cultured with or without the basal lamina extract, Matrigel. Within each group OEC were cultured in the presence of either estradiol-17 beta or a carrier control. All groups were subcultured three times. Epithelial cell morphology and fu...
Roberto da Costa RP, Costa AS, Korzekwa AJ, Platek R, Siemieniuch M, Galvão A, Redmer DA, Silva JR, Skarzynski DJ, Ferreira-Dias G.Nitric oxide (NO) plays an important role in prostaglandin secretion and angiogenesis in the reproductive system. In the present study, the roles of the NO donor spermine NONOate and tumour necrosis factor-alpha (TNF; as a positive control) in prostaglandin production and angiogenic activity of equine endometria during the oestrous cycle were evaluated. In addition, the correlation between NO production and the expression of key prostaglandin synthase proteins was determined. The protein expression of prostaglandin F synthase (PGFS) increased in early and mid-luteal stages, whereas that of pro...
Kadivar A, Shams Esfandabadi N, Dehghani Nazhvani E, Shirazi A, Ahmadi E.Protamines substitute DNA-binding histones during late spermatogenesis in sperm nucleus. Stallion sperm contains all three variants of these arginine-rich and positively charged nuclear proteins (P1, P2 and P3). Two variants of protamine-2, that is P2 and P3, constitute approximately 15% of the entire protamine content. Also, the ratio of protamine-1 to protamine-2 varies among different mammalian species, and abnormal protamine ratios and protamine content are correlated with male infertility. In this study, changes in protamine mRNA abundance for all three protamines were investigated in sta...
Hansen N, Buchner HH, Haller J, Windischbauer G.To evaluate using strain gauges, a hoof cast with heel wedge, and a therapeutic shoe with unsupported toe for their effectiveness in redistribution of load from the dorsal hoof wall. Methods: In vitro biomechanical study. Methods: Twenty forelimb specimens. Methods: Rosette strain gauges were placed on the dorsal and lateral hoof wall of 20 normal shaped hooves. Limbs were loaded vertically using a tensile testing machine with a 1 Hz sinusoidally cycling load up to 3000 N during 15 seconds. Mean values of principal strain and direction at 2500 N load were calculated for 3 experimental conditio...
Bruemmert JE, Coy RC, Squires EL, Graham JK.Stallion spermatozoa maintain high fertilizing capacity if cooled to 5 degrees C and inseminated within 24 h. However, if spermatozoa are stored for 48 h, fertilizing capacity declines. Therefore, multiple shipments of semen are often required to inseminate mares that remain in estrus for days. Therefore, experiments were designed to determine if adding antioxidants to stallion spermatozoa stored at 5 degrees C for 48 h could maintain motility and fertilizing ability. In the first experiment stallion spermatozoa were incubated in a skim milk (SM) or a skim milk-egg yolk medium in combination w...
Heneine LG, Carvalho AD, Barbosa CF, Arávjo dos Santos MR.The objective of this study was the search for a suitable venom antigen to be used in an in vitro alternative immunoassay, to the standard antivenom neutralization assay using mice. Bothrops jararaca venom was fractionated in DEAE-Sephacel columns and the fractions were tested for a correlation between antibody capture enzyme linked immunosorbent assay (ELISA) absorbance values and the 'in vivo' antivenom potency. Individual antivenoms from 14 horses and 15 separate FUNED polyspecific Bothrops ampouled antivenoms (final product) were used. Fractions showing the higher correlations were further...
Patterson Rosa L, Walker N, Mallicote M, MacKay RJ, Brooks SA.Similar to humans, the horse relies predominantly on the evaporation of sweat from the skin surface to dissipate excess body heat. Loss of the sweat response or anhidrosis can result in life-threatening hyperthermia. Anhidrosis occurs more frequently in some breeds as well as occurs at an increased frequency among individuals with a family history, suggesting a heritable component to the pathology. Given the natural occurrence and indications of genetic components in the etiology, we utilized genomics to better understand the molecular mechanisms involved in sweat response. We performed a case...
Ryan C, Giguère S, Hagen J, Hartnett C, Kalyuzhny AE.Peripheral blood mononuclear cells (PBMCs) were obtained from 6 foals <1 week of age, 6 foals between 3 and 4 months of age, and 10 adult horses. PBMCs were stimulated with concanavalin A (ConA) or calcium ionomycin-phorbol myristate acetate (CaI-PMA) and the frequency of interferon IFN-gamma and IL-4 secreting cells was measured using an equine-specific ELISPOT assay. The number of IFN-gamma secreting cells was significantly lower in both groups of foals than in adult horses regardless of the mitogen used for stimulation. The number of IFN-gamma secreting cells was significantly higher in ...
Smits K, Govaere J, Hoogewijs M, Piepers S, Van Soom A.Intracytoplasmic sperm injection (ICSI) is the method of choice for the in vitro production (IVP) of equine embryos. However, conventional ICSI has been associated with mechanical damage to the oocyte caused by the deformation of the zona pellucida (ZP) and exposure of the oolemma to negative pressure during injection. Introduction of the less traumatic and more efficient piezo drill-assisted ICSI (PDAI) yielded higher cleavage rates and more consistent results. Nevertheless, PDAI is also associated with disadvantages such as the use of mercury and possible DNA damage. This led us to explore a...
Racine T, Denizot M, Pannetier D, Nguyen L, Pasquier A, Raoul H, Saluzzo JF, Kobinger G, Veas F, Herbreteau CH.There is no vaccine or approved therapy against lethal Ebola virus (EBOV). We investigated a proven technology platform to produce polyclonal IgG fragments, F(ab')2, against EBOV. Horses immunized with nanoparticles harboring surface glycoprotein trimers of EBOV-Zaire/Makona produced anti-Ebola IgG polyclonal antibodies with high neutralization activity. Highly purified equine anti-Ebola F(ab')2 showed strong cross-neutralization of 2 Zaire EBOV strains (Gabon 2001 and Makona) and in vivo 3 or 5 daily F(ab')2 intraperitoneal injections provided 100% protection to BALB/c mice against lethal EBO...
Lentz LR, Valberg SJ, Mickelson JR, Gallant EM.To determine whether increased sensitivity to pharmacologic agents was a general property of equine exertional myopathies, including polysaccharide storage myopathy (PSSM) in Quarter Horses. Methods: 5 adult Quarter Horses with exertional rhabdomyolysis and abnormal polysaccharide accumulation in skeletal muscle and 4 clinically normal adult Quarter or Quarter-type horses. Methods: Twitch time course measurements and contracture responses to various concentrations of caffeine and halothane for small bundles of intact external intercostal muscle fibers were measured in all horses. Results: Caff...
Peroni JF, Harrison WE, Moore JN, Graves JE, Lewis SJ, Krunkosky TM, Robertson TP.Equine laminitis purportedly involves haemodynamic dysfunction at the level of the laminar vasculature. However, to date, no studies have been performed characterising the function of laminar arteries and veins during the prodromal stages of equine laminitis. Objective: That the prodromal stages of laminitis are associated with contractile dysfunction of the equine laminar vasculature. Objective: To assess contractile function of laminar arteries and veins to phenylephrine (PE) and 5-hydroxytryptamine (5-HT). Methods: Horses were administered black walnut heartwood extract (BWHE) or water (con...
Underwood C, Collins SN, van Eps AW, Allavena RE, Medina-Torres CE, Pollitt CC.There are no experimentally validated pharmacological means of preventing laminitis; however, locally acting pharmaceutical agents with the potential to prevent laminitis have been identified. Demonstrating therapeutic drug concentrations in lamellar tissue is essential for evaluating the efficacy of these agents. The aim of this study was to develop an experimental technique for repeatedly sampling lamellar interstitial fluid. A technique for placing ultrafiltration probes was developed in vitro using 15 cadaver limbs. Subsequently, lamellar ultrafiltration probes were placed in one forelimb ...
Mudge MC, Macdonald MH, Owens SD, Tablin F.To compare viability of equine whole blood stored by 4 different methods, and to establish optimal storage protocols for an equine autologous blood donation program. Methods: In vitro study of stored equine whole blood. Animals- Six healthy adult horses. Methods: Blood from each horse was collected into 4 different containers: glass bottles containing acid-citrate-dextrose solution (ACD), plastic bags containing ACD, citrate-phosphate-dextrose (CPD), and CPD with supplemental adenine (CPDA-1). Blood was stored for 5 weeks and sampled at 2-day intervals. Standard hematologic and biochemical var...
Bowen IM, Marr CM, Chester AH, Wheeler-Jones CP, Elliott J.The equine aortic valve is subject to non-inflammatory degenerative changes, associated with aortic valvular regurgitation (AR). This disease shares pathological and epidemiological features with AR in humans, and may serve as a useful model to study in-vitro functional responses associated with aging and disease. The study aim was to determine the contractile properties of the normal equine aortic valve. Methods: The contractile responses of equine aortic valves to angiotensin II, the thromboxane-mimetic U44069, endothelin-1, 5-hydroxytryptamine and the alpha-adrenoceptor agonists medetomidin...
Morrell JM, Geraghty RM.A method of removing equine arteritis virus (EAV) from equine semen used for artificial insemination is urgently needed. Recent medical studies suggest that a double semen processing technique of density gradient centrifugation followed by a 'swim-up' can provide virus-free sperm preparations for assisted reproduction. Objective: To investigate the use of the double semen processing technique to obtain virus-free sperm preparations from stallion semen containing EAV. Methods: Aliquots of an ejaculate from an uninfected stallion were spiked with virus and processed by the double processing tech...
Raza F, Babasyan S, Larson EM, Freer HS, Schnabel CL, Wagner B.Interleukin-4 (IL-4) is a key cytokine secreted by type 2 T helper (Th2) cells that orchestrates immune responses during allergic reactions. Human and mouse studies additionally suggest that basophils have a unique role in the regulation of allergic diseases by providing initial IL-4 to drive T cell development towards the Th2 phenotype. Equine Culicoides hypersensitivity (CH) is a seasonal immunoglobulin E (IgE)-mediated allergic dermatitis in horses in response to salivary allergens from Culicoides (Cul) midges. Here, we analyzed IL-4 production in peripheral blood mononuclear cells (PBMC) o...
Pechanec MY, Beall JM, Katzman S, Maga EA, Mienaltowski MJ.Tendinopathies remain the leading contributor to career-ending injuries in horses because of the complexity of tendon repair. As such, cell-based therapies like injections of adipose-derived mesenchymal stem cells (ADMSCs, or MSCs) into injured tendons are becoming increasingly popular though their long-term efficacy on a molecular and wholistic level remains contentious. Thus, we co-cultured equine MSCs with intrinsic (tendon proper) and extrinsic (peritenon) tendon cell populations to examine interactions between these cells. Gene expression for common tenogenic, perivascular, and differenti...
