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Topic:Experimental Methods

Experimental methods in equine research encompass a variety of techniques and procedures used to investigate physiological, behavioral, and health-related aspects of horses. These methods are designed to collect data that can enhance the understanding of equine biology and inform veterinary practices. Common experimental approaches include controlled trials, observational studies, and laboratory-based experiments. Techniques may involve the use of non-invasive imaging, biochemical assays, and physiological monitoring to assess parameters such as metabolic function, locomotion, and disease progression. This page compiles peer-reviewed research studies and scholarly articles that explore the application, development, and evaluation of experimental methods in the study of equine science.
Development and characterization of an equine skin-equivalent model.
Veterinary dermatology    July 18, 2014   Volume 25, Issue 5 475-e77 doi: 10.1111/vde.12134
Cerrato S, Ramió-Lluch L, Brazís P, Rabanal RM, Fondevila D, Puigdemont A.There is increasing interest in the biological and pathological study of equine skin owing to the high prevalence of cutaneous diseases in horses. However, knowledge of equine skin cell biology and cultures is limited by the low number of in vitro studies in the literature. Objective: The objective of the study was to develop and characterize an in vitro equine skin equivalent. Methods: Cultures of pure equine keratinocytes and dermal fibroblasts were obtained by enzymatic digestion of skin biopsies. Fibroblasts were embedded into type I collagen matrices to obtain dermal scaffolds, the surfac...
Therapeutic potential of Pirfenidone for treating equine corneal scarring.
Veterinary ophthalmology    July 15, 2014   Volume 18, Issue 3 242-250 doi: 10.1111/vop.12194
Fink MK, Giuliano EA, Tandon A, Mohan RR.To evaluate the safety and efficacy of Pirfenidone (PFD) in the treatment of equine corneal fibrosis using an in vitro model. Methods: Healthy donor equine corneas were collected and used to generate primary equine corneal fibroblasts (ECFs) by growing cultures in minimal essential medium supplemented with 10% fetal bovine serum. Equine corneal myofibroblasts (ECMs), used as a model of equine corneal fibrosis, were produced by growing ECF cultures in serum-free medium containing transforming growth factor β1 (1 ng/mL). Trypan blue viability assays and changes in ECF morphology were utilized t...
Clinical observations on the course of oxytocin- or prostaglandin E2/oxytocin-induced parturition in mares.
Polish journal of veterinary sciences    July 6, 2014   Volume 17, Issue 2 347-351 doi: 10.2478/pjvs-2014-0047
Witkowski M, Witkowski M, Pawłowski K.The objective of this study was to compare the course of parturitions induced with sole oxytocin with those induced with the combination of intracervical prostaglandin E2 jelly and oxytocin. For this purpose 13 mares in advanced pregnancy were allocated to the groups pretreated with either intracervical PGE2 (experimental group) or saline (control group) two hours before intravenous oxytocin (5 IU) administration. The mares were compared with respect to cervical dilation diameter (CDD) 20 min. after oxytocin injection. Time intervals from the first oxytocin dose to: the first external signs of...
Comparison of respiratory function during TIVA and isoflurane anaesthesia in ponies Part II: breathing patterns and transdiaphragmatic pressure.
Veterinary anaesthesia and analgesia    July 2, 2014   Volume 41, Issue 6 592-601 doi: 10.1111/vaa.12151
Kowalczyk L, Steblaj B, Schauvliege S, Schramel JP, Pavlidou K, Savvas I, Duchateau L, Gasthuys F, Moens Y.To compare breathing patterns and transdiaphragmatic pressure during total intravenous (TIVA) and isoflurane anaesthesia in ponies. Methods: Experimental, cross-over study. Methods: Six healthy ponies weighing 286 (233-388) ± 61 kg, age 13 (9-16) ± 3 years. Methods: Following premedication with romifidine [80 μg kg(-1) intravenously (IV)], general anaesthesia was induced with midazolam (0.06 mg kg(-1) IV) and ketamine (2.5 mg kg(-1) IV) and maintained with either isoflurane (Fe'Iso = 1.1%) (T-ISO) or an IV combination of romifidine (120 μg kg(-1) per hour), midazolam (0.09 mg kg(-1) hour(-...
The recent European isolate (08P178) of equine arteritis virus causes inflammation but not arteritis in experimentally infected ponies.
Journal of comparative pathology    June 27, 2014   Volume 151, Issue 2-3 238-243 doi: 10.1016/j.jcpa.2014.04.008
Vairo S, Saey V, Bombardi C, Ducatelle R, Nauwynck H.In the last two decades, outbreaks of equine viral arteritis (EVA) have been reported in Europe, but little is known about these European isolates of equine arteritis virus (EAV). EAV European strain (08P178, EU-1 clade) isolated from one of these recent outbreaks is able to cause clinical signs on experimental infection. The aim of the present study was to investigate the microscopical lesions induced by this isolate after experimental infection of ponies. Animals were killed at 3, 7, 14 and 28 days post infection (dpi). At 3 dpi, lesions were essentially restricted to the respiratory tract a...
The use of the noninferiority analysis in clinical studies.
Equine veterinary journal    June 10, 2014   Volume 46, Issue 4 399-401 doi: 10.1111/evj.12268
Bermingham EC, del Castillo JR, Radecki SV.No abstract available
In vitro mechanical testing of braided polyurethane elastic fiber and braided polyester for equine laryngoplasty.
Veterinary surgery : VS    June 5, 2014   Volume 44, Issue 2 223-230 doi: 10.1111/j.1532-950X.2014.12184.x
Willsallen H, Heller J, Kark L, Hilbert BJ.In vitro comparison of the mechanical properties of braided polyurethane elastomer (Lycra®) and braided polyester (Ethibond™) (1) when inserted into the muscular process of the arytenoid cartilage and (2) as suture loops. Methods: Experimental. Methods: Equine cadaver larynges (n = 15). Methods: The muscular processes (n = 30) of the arytenoid cartilages were dissected from each larynx and embedded in a resin base. Lycra® and Ethibond™ prostheses were randomly allocated to the left or right muscular process and each underwent cyclic fatigue (25-50 N) followed by load-to-failure...
Challenges of thermal nociceptive threshold testing in the donkey.
Veterinary anaesthesia and analgesia    May 29, 2014   Volume 42, Issue 2 205-214 doi: 10.1111/vaa.12182
Grint NJ, Whay HR, Beths T, Yvorchuk K, Murrell JC.To evaluate a thermal nociceptive threshold (TNT) testing device in the donkey, and the influence of potential confounding factors on TNTs. Methods: Two groups (Group 1 and Group 2) of eight castrated male donkeys aged 4-9 years, weighing 105-170 kg. Methods: TNTs were measured by heating a thermal probe on skin until an end-point behaviour (threshold temperature) or a cut-out temperature (51 °C) was reached. The withers and the dorsal aspect of the distal limb were used as sites for TNT testing. The effects on TNT of different confounding factors: the limb tested; rate of heating; and amb...
Comparison of cardiac output determined by an ultrasound velocity dilution cardiac output method and by the lithium dilution cardiac output method in juvenile horses with experimentally induced hypovolemia.
American journal of veterinary research    May 29, 2014   Volume 75, Issue 6 565-571 doi: 10.2460/ajvr.75.6.565
Shih AC, Queiroz P, Vigani A, Da Cunha A, Pariaut R, Ricco C, Bornkamp J, Garcia-Pereira F, Bandt C.To assess the accuracy of an ultrasound velocity dilution cardiac output (UDCO) method, compared with that of the lithium dilution cardiac output (LiDCO) method, for determination of cardiac output (CO) in juvenile horses with experimentally induced hypovolemia. Methods: 12 anesthetized 2- to 6-month-old horses. Methods: For each anesthetized horse, CO was determined by the LiDCO and UDCO methods prior to any intervention (baseline state), after withdrawal of approximately 40% of the horse's blood volume (low CO state), after maintenance of hypovolemia and infusion of norepinephrine until mean...
Influence of xylazine on the function of the LiDCO sensor in isoflurane anaesthetized horses.
Veterinary anaesthesia and analgesia    May 29, 2014   Volume 42, Issue 2 142-149 doi: 10.1111/vaa.12181
Hopster K, Ambrisko TD, Stahl J, Schramel JP, Kästner SB.Previous studies showed an influence of xylazine on the LiDCO sensor in vitro and in standing horses, but did not prove that this interaction caused error in LiDCO measurements. Therefore, agreement of cardiac output (CO) measurements by LiDCO and bolus-thermodilution (BTD) was determined in horses receiving xylazine infusions. Methods: Prospective, experimental study. Methods: Eight Warmblood horses. Methods: All horses were premedicated with xylazine. Anaesthesia was induced with midazolam and ketamine and was maintained with isoflurane in oxygen. During six hours of anaesthesia CO measureme...
Effectiveness of conventional and hydrosurgical debridement methods in reducing Staphylococcus aureus inoculation of equine muscle in vitro.
Equine veterinary journal    May 26, 2014   Volume 47, Issue 2 218-222 doi: 10.1111/evj.12284
Skärlina EM, Wilmink JM, Fall N, Gorvy DA.The success of primary healing of equine traumatic wounds is dependent on thorough debridement. A specific hydrosurgical debridement device (Versajet(TM) )(a) is gentle to viable tissues, yet effectively removes macroscopic contaminants and debris. We wished to investigate whether it is effective in reducing bacterial burden and whether it differs from traditional methods. No previous reports compare hydrosurgical debridement and conventional wound debridement with regard to bacterial reduction from in vitro inoculated soft tissue. Objective: To assess the effectiveness of hydrosurgical debrid...
Surgical stress influences cytokine content in autologous conditioned serum.
Equine veterinary journal    May 26, 2014   Volume 47, Issue 2 212-217 doi: 10.1111/evj.12277
Fjordbakk CT, Johansen GM, Løvås AC, Oppegård KL, Storset AK.No recommendations have been made regarding the relative timing of blood collection for autologous conditioned serum (ACS) preparation and surgical procedures. Objective: 1) To identify effects of surgical stress on cytokine levels in ACS, 2) identify haematological markers for prediction of cytokine production in ACS and 3) investigate the necessity for specialised ACS containers when preparing a cytokine-rich serum. Methods: Experimental in vitro study. Methods: Blood was drawn from 15 stallions admitted for elective castration preoperatively and 22-24 h post operatively and incubated in A...
Comparative study of the effects of fetal bovine serum versus horse serum on growth and differentiation of primary equine bronchial fibroblasts.
BMC veterinary research    May 26, 2014   Volume 10 119 doi: 10.1186/1746-6148-10-119
Franke J, Abs V, Zizzadoro C, Abraham G.Airway fibroblasts have become a critical addition to all facets of structural lung tissue changes such as in human asthma and chronic obstructive pulmonary disease, but little is known about their role in the equine recurrent airway obstruction, a disease that resembles to the human asthma. Since the equine bronchial fibroblasts (EBF) have not been isolated and characterized yet, the use of defined medium was investigated. Results: Primary EBF were cultured on non-collagen coated flasks without serum or in the presence of fetal bovine serum (FBS) or horse serum (HS) or in serum depleted mediu...
Shetland ponies (Equus caballus) show quantity discrimination in a matching-to-sample design.
Animal cognition    May 16, 2014   Volume 17, Issue 6 1233-1243 doi: 10.1007/s10071-014-0753-0
Gabor V, Gerken M.Numerical competence is one of the aspects of animal cognition with a long history of research interest, but few results are available for the horse. In the present study, we investigated the ability of three Shetland ponies to discriminate between different quantities of geometric symbols presented on a computer screen in a matching-to-sample arrangement. In Experiment 1, the ponies had to relate two similar quantities to another, paired in contrasts (1 vs. 2, 3 vs. 4 and 4 vs. 5) of the same stimulus (dot). Specific pairs of quantities (all differing by one) of up to five different geometric...
Treatment of experimentally induced osteoarthritis in horses using an intravenous combination of sodium pentosan polysulfate, N-acetyl glucosamine, and sodium hyaluronan.
Veterinary surgery : VS    May 13, 2014   Volume 43, Issue 5 612-622 doi: 10.1111/j.1532-950X.2014.12203.x
Koenig TJ, Dart AJ, McIlwraith CW, Horadagoda N, Bell RJ, Perkins N, Dart C, Krockenberger M, Jeffcott LB, Little CB.To assess the effects of sodium pentosan polysulfate (PPS), N-acetyl glucosamine (NAG), and sodium hyaluronan (HA) in horses with induced osteoarthritis (OA). Methods: Experimental. Methods: Adult Standard bred horses (n = 16). Methods: OA was induced arthroscopically in 1 intercarpal joint; 8 horses were administered 3 mg/kg PPS, 4.8 mg/kg NAG, and 0.12 mg/kg HA (PGH), intravenously (IV), weekly and 8 horses were administered an equivalent volume of saline IV until study completion (day 70). Horses underwent a standardized treadmill exercise program. Clinical and radiographic findin...
Efficacy of a pectin-lecithin complex for treatment and prevention of gastric ulcers in horses.
The Veterinary record    May 12, 2014   Volume 175, Issue 6 147 doi: 10.1136/vr.102359
Sanz MG, Viljoen A, Saulez MN, Olorunju S, Andrews FM.The objective of this study was to evaluate the effect of a commercial feed supplement containing pectin-lecithin on squamous mucosa ulceration in horses exposed to an experimental ulceration model. Five mares were treated while five mares were controls for this crossover, blinded study. The mares were fed concentrates and hay and were stabled with a two-hour turn out per day for a period of four weeks. The pectin-lecithin complex was fed for the duration of the study on the treated group. At the end of a four-week period, all mares underwent a seven-day alternating feed deprivation (week 5). ...
Ultrafiltration of equine digital lamellar tissue.
Veterinary journal (London, England : 1997)    May 9, 2014   Volume 202, Issue 2 314-322 doi: 10.1016/j.tvjl.2014.05.007
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 ...
A new method of producing casts for anatomical studies.
Anatomical science international    May 1, 2014   Volume 89, Issue 4 255-265 doi: 10.1007/s12565-014-0240-3
De Sordi N, Bombardi C, Chiocchetti R, Clavenzani P, Trerè C, Canova M, Grandis A.The objective of the present study was to verify if polyurethane foam is a suitable material to make accurate casts of vessels and viscera, and to develop a method based on its use for anatomical studies. This new technique has been tested primarily on the lungs of different animals, but also on the renal, intestinal and equine digital vessels. It consisted of three steps: specimen preparation, injection of the foam and corrosion of the cast. All structures injected with foam were properly filled. The bronchial tree and the vessels could be observed up to their finer branches. The method is in...
A comparison of the histological structure of the placenta in experimental animals.
Journal of toxicologic pathology    April 30, 2014   Volume 27, Issue 1 11-18 doi: 10.1293/tox.2013-0060
Furukawa S, Kuroda Y, Sugiyama A.The primary function of the placenta is to act as an interface between the dam and fetus. The anatomic structure of the chorioallantoic placenta in eutherian mammals varies between different animal species. The placental types in eutherian mammals are classified from various standpoints based on the gross shape, the histological structure of the materno-fetal interface, the type of materno-fetal interdigitation, etc. Particularly, the histological structure is generally considered one of the most useful and instructive classifications for functionally describing placental type. In this system,...
Mechanical properties of various suture materials and placement patterns tested with surrogate in vitro model constructs simulating laryngeal advancement tie-forward procedures in horses.
American journal of veterinary research    April 26, 2014   Volume 75, Issue 5 500-506 doi: 10.2460/ajvr.75.5.500
Santos MP, Gutierrez-Nibeyro SD, Horn GP, Johnson AJ, Stewart MC, Schaeffer DJ.To compare the mechanical properties of laryngeal tie-forward (LTF) surrogate constructs prepared with steel fixtures and No. 5 braided polyester or braided polyethylene by use of a standard or a modified suture placement technique. Methods: 32 LTF surrogate constructs. Methods: Surrogate constructs were prepared with steel fixtures and sutures (polyester or polyethylene) by use of a standard or modified suture placement technique. Constructs underwent single-load-to-failure testing. Maximal load at failure, elongation at failure, stiffness, and suture breakage sites were compared among constr...
Assessing the effectiveness of 30% sodium chloride aqueous solution for the preservation of fixed anatomical specimens: a 5-year follow-up study.
Journal of anatomy    April 25, 2014   Volume 225, Issue 1 118-121 doi: 10.1111/joa.12185
de Oliveira FS.Anatomical specimens used in human or veterinary anatomy laboratories are usually prepared with formaldehyde (a cancerous and teratogenic substance), glycerin (an expensive and viscous fluid), or ethanol (which is flammable). This research aimed to verify the viability of an aqueous 30% sodium chloride solution for preservation of anatomical specimens previously fixed with formaldehyde. Anatomical specimens of ruminant, carnivorous, equine, swine and birds were used. All were previously fixed with an aqueous 20% formaldehyde solution and held for 7 days in a 10% aqueous solution of the same ...
In vivo diffusion characteristics following perineural injection of the deep branch of the lateral plantar nerve with mepivacaine or iohexol in horses.
Equine veterinary journal    April 20, 2014   Volume 47, Issue 2 230-234 doi: 10.1111/evj.12261
Contino EK, King MR, Valdés-Martínez A, McIlwraith CW.Hindlimb proximal suspensory desmopathy is a common injury of sport horses but diagnosis can be difficult because diagnostic analgesia of the region lacks specificity. Perineural analgesia of the deep branch of the lateral plantar nerve (DBLPN) has been proposed as a more specific method of isolating pain of the proximal aspect of the suspensory ligament but the technique has not been evaluated in vivo. Objective: To determine the extent of diffusion of contrast medium and mepivacaine following DBLPN analgesia using a single-needle injection technique and to determine if there is inadvertent i...
Ultrasound-guided injection of the median artery in the standing sedated horse.
Equine veterinary journal    April 20, 2014   Volume 47, Issue 2 245-248 doi: 10.1111/evj.12260
Spriet M, Trela JM, Galuppo LD.Injection of the median artery of horses leads to better distribution and persistence of mesenchymal stem cells than i.v. regional limb perfusion. Due to technical difficulties, intra-arterial injections thus far have only been performed under general anaesthesia. Objective: To assess the feasibility of injection of the median artery in standing sedated horses. Methods: Experimental study. Methods: Six horses were included in the study. After median and ulnar regional analgesia, radiographic contrast material was injected in the median artery of both front limbs, using a catheter in one limb a...
Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study.
PeerJ    April 17, 2014   Volume 2 e353 doi: 10.7717/peerj.353
Warnock JJ, Fox DB, Stoker AM, Beatty M, Cockrell M, Janicek JC, Cook JL.Introduction. Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM) formation of equine fibroblast-like synoviocytes (FLS) cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-l...
Swing phase kinematics of horses trotting over poles.
Equine veterinary journal    April 9, 2014   Volume 47, Issue 1 107-112 doi: 10.1111/evj.12253
Brown S, Stubbs NC, Kaiser LJ, Lavagnino M, Clayton HM.Trotting over poles is used therapeutically to restore full ranges of limb joint motion. The mechanics of trotting over poles have not yet been described, hence quantitative evidence for the presumed therapeutic effects is lacking. Objective: To compare limb kinematics in horses trotting over level ground, over low poles and over high poles to determine changes in joint angulations and hoof flight arcs. Methods: Repeated measures experimental study in sound horses. Methods: Standard motion analysis procedures with skin-fixed reflective markers were used to measure swing phase kinematics from 8...
Comparison of 2 equine transfixation pin casts and the effects of pin removal.
Veterinary surgery : VS    April 7, 2014   Volume 43, Issue 4 430-436 doi: 10.1111/j.1532-950X.2014.12182.x
Williams JM, Elce YA, Litsky AS.To (1) compare strain on the dorsal aspect of the proximal phalanx (P1) between 2 types of transfixation pin casts (TPC) and (2) evaluate the change in strain as the pins are removed. Methods: Experimental. Methods: Equine cadaver forelimbs (n = 10 pair). Methods: Each limb of a pair was assigned to 1 of 2 TPC constructs. Construct 1 consisted of a TPC with 2 positive profile, centrally threaded pins placed in the distal aspect of the third metacarpus (MC3) and construct 2 had 4 smooth Steinmann pins placed similarly. A strain gauge was mounted on P1 and axial compression (444.8-5337.9 N) appl...
An ex vivo model to evaluate the effect of cyclical adductory forces on maintenance of arytenoid abduction after prosthetic laryngoplasty performed with and without mechanical arytenoid abduction.
Veterinary surgery : VS    April 5, 2014   Volume 43, Issue 5 598-605 doi: 10.1111/j.1532-950X.2014.12178.x
McClellan NR, Santschi EM, Hurcombe SD, Litsky AS.To (1) develop a model of cyclical adduction force on an abducted left arytenoid cartilage that mimics swallowing or coughing; (2) determine if arytenoid abduction by a clamp before knot tying will improve the maintenance of abduction under cyclical adduction testing. Methods: Experimental. Methods: Cadaveric equine larynges (n = 14). Methods: Left laryngoplasty performed using a single suture of #5 Ethibond with (n = 7) and without (n = 7) abducting the arytenoid with a clamp before knot tying. Each laryngoplasty was loaded cyclically from 2 to 26 N at 0.5 Hz for 5000 cycles i...
Biomechanical testing of a novel tendon implant device for the repair of equine flexor tendon lacerations.
Veterinary surgery : VS    April 5, 2014   Volume 43, Issue 6 685-690 doi: 10.1111/j.1532-950X.2014.12181.x
Barrett EJ, Munsterman AS, Hanson RR, Jackson RL.To compare in vitro strength and failure characteristics of a tendon implant against a 3 loop pulley suture pattern for equine superficial digital flexor tendon (SDFT) laceration repair. Methods: Experimental. Methods: Cadaveric equine forelimb SDFT (n = 16). Methods: One tendon of each of 8 pair of SDFT was randomly selected for repair with either the 3-loop pulley (3LP) suture pattern using 2 polydioxanone or with 4 stainless steel suture and anchor implants (SA). Ultimate load to failure, mode of failure, gap at failure, and load to 2 mm gap were obtained using a materials testing sys...
Attempts to induce nocardioform placentitis (Crossiela equi) experimentally in mares.
Equine veterinary journal    April 1, 2014   Volume 47, Issue 1 91-95 doi: 10.1111/evj.12249
Canisso IF, Ball BA, Erol E, Claes A, Scoggin KE, McDowell KJ, Williams NM, Dorton AR, Wolfsdorf KE, Squires EL, Troedsson MH.Nocardioform placentitis in horses is poorly understood, and the development of an experimental model would be of help in understanding the pathogenesis of the disease. Objective: To investigate whether (1) intrauterine inoculation of Crossiela equi during the periovulatory period or (2) i.v., oral or intranasopharyngeal inoculation of C. equi during midgestation would result in nocardioform placentitis, and (3) before and after mating endometrial swabs present evidence of nocardioform placentitis-associated organisms (C. equi or Amycolatopsis spp.). Methods: In Study I, mares (n = 20) rec...
Evaluation of the effect of onlay mesenteric flaps on end-to-end jejunojejunostomy healing in horses.
Veterinary surgery : VS    April 1, 2014   Volume 43, Issue 4 479-486 doi: 10.1111/j.1532-950X.2014.12120.x
Aristizabal FA, Lopes MA, Silva A, Avanza MF, Nieto JE.To evaluate the effect of onlay mesenteric flaps (MFs) with end-to-end jejunojejunostomy on stomal diameter, length of jejunum with reduced stomal diameter, abdominal adhesion formation, and healing. Methods: Experimental study. Methods: Healthy adult horses (n = 6). Methods: Two hand sewn end-to-end jejunal anastomoses using a 1 layer simple continuous serosubmucosal suture pattern were performed in each horse ∼ 2 and 5 m oral to the ileocecal fold. Using a random design for selection anastomosis location (oral or aboral), 1 anastomosis was covered with 2 adjacent onlay MFs secured with int...
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