We improved a method of chronic duodenal cannulation to study intestinal transport of solutes in an in vivo model (pigeon, Columba livia). A hypoallergenic cannula was inserted into the proximal part of the small intestine of pigeons and used for solution administration. Recovery from surgery was extremely rapid and animals started eating and drinking within a day. After surgery, the body mass of cannulated pigeons was stable, and no adverse effects in the weight could be detected. The method is simple, economical and useful to determine intestinal bioavailability of solutes, for nutritional and ecological studies, in intact animals without influence of anesthesia.Fil: Chediack, Juan Gabriel. Universidad Nacional de San Luis. Facultad de Ciencias Humanas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Quimica, Bioquimica y Farmacia. Departamento de Bioquímica y Ciencias Biológicas; ArgentinaFil: Cid, Fabricio Damian. Universidad Nacional de San Luis. Facultad de Ciencias Humanas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Quimica, Bioquimica y Farmacia. Departamento de Bioquímica y Ciencias Biológicas; ArgentinaFil: Fasulo, S. V.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Ciencias Humanas; ArgentinaFil: Caviedes Vidal, Enrique Juan Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Ciencias Humanas; Argentina. Universidad Nacional de San Luis. Facultad de Quimica, Bioquimica y Farmacia. Departamento de Bioquímica y Ciencias Biológicas; Argentin

Caviedes Vidal, Enrique Juan Raul

Chediack, Juan Gabriel

Cid, Fabricio Damian

Fasulo, S. V.

Veterinární Medicína

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Veterinarni Medicina, 55, 2010 (8): 383–388 Original Paper383Chronic cannulation in the small intestine of feral pigeons (Columba livia) to assess bioavailabilityJ.G. Chediack1,2,3, F.D. Cid1,2,3, S.V. Fasulo1,2, E. Caviedes-Vidal1,2,31Laboratory of Biology “Prof. E. Caviedes Codelia”, Facultad de Ciencias Humanas, Universidad Nacional de San Luis, San Luis, Argentina2Laboratory of Integrative Biology, Institute for Multidisciplinary Research in Biology (IMIBIO-SL), Consejo Nacional de Investigaciones Científicas y Técnicas, San Luis, Argentina3Dept. Biochemistry and Biological Sciences, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, San Luis, ArgentinaABSTRACT: We improved a method of chronic duodenal cannulation to study intestinal transport of solutes in an in vivo model (pigeon, Columba livia). A hypoallergenic cannula was inserted into the proximal part of the small intestine of pigeons and used for solution administration. Recovery from surgery was extremely rapid and animals started eating and drinking within a day. After surgery, the body mass of cannulated pigeons was stable, and no adverse effects in the weight could be detected. The method is simple, economical and useful to determine intestinal bioavailability of solutes, for nutritional and ecological studies, in intact animals without influence of anesthesia.Keywords: chronic cannulation; small intestine; pigeons; bioavailabilityThe study was supported by FONCYT 7-7488 and UNSL-CyT 22Q751 grants to EC-V, and PIP No. 11220090100998 CONICET and UNSL-CyT No. 0110 grants to JGC.The study of intestinal absorption is important for research in areas as diverse as clinical medicine, nutrition and ecotoxicology. In the first case, sev-eral studies have evaluated the passive permeability characteristics of the small intestine using a series of nonelectrolyte hydrosoluble probes, and they are routinely used in oral absorption studies to test for intestinal pathologies (Elia et al., 1987; Bjarnason et al., 1995). In the second and third cases, intestinal absorption is relevant for the different pathways of nutrient absorption and their regulation by dif-ferent factors, because intestinal permeability is a major determinant of the oral bioavailability of hydrosoluble nutrients and toxins (Caviedes-Vidal, 2003; Chediack et al., 2003; Cid et al., 2005).Many techniques have been developed for in vitro, in situ and in vivo studies of intestinal ab-sorption processes. To understand the absorption process of a compound, a proper method has to be chosen. The in vitro methods to study intestinal absorption are: cell lines (e.g. Caco-2, HT-29 and MDKC), everted sleeves, brush border membrane vesicles and Ussing chambers. The in situ method involves perfusion of an intestinal segment, fol-lowed by recirculation of the substrate through the intestinal segment and the determination of the amount of substrate remaining in the solution (Roig and Vinardell, 1990; Perez et al., 1993). There are two main advantages to measuring absorption in the intact animal (in vivo) over in anesthetized animals (in situ) or by tissue preparations. First, while the latter methods can demonstrate condi-tions and mechanisms by which a process can oc-cur, they cannot by themselves demonstrate the mechanisms by which it normally happens. Second, there is evidence that approaches not relying on intact animals may introduce artifacts in intestinal absorption (Uhing and Kimura, 1995).Original Paper Veterinarni Medicina, 55, 2010 (8): 383–388384In this study we propose the use, for the first time, of another technique to measure in vivo intestinal bioavailability, the needle catheter duodenostomy. The duodenostomy is a surgical procedure where a feeding tube is inserted into the duodenum. In birds, it is a viable method of alimentation (Goring et al., 1986). We suggest the exploitation of this technique, developed in the beginning for duodenal alimentation, to assess bioavailability by pharma-cokinetic techniques. The classical pharmacoki-netic technique involves feeding (by gavage) and injecting probes and then measuring those probes in the blood at various times post-gavage or post-injection (Chediack et al., 2001). Studies of intes-tinal transport have historically been based on a variety of in vitro and in vivo methods, which in turn allows the assembling of a picture of how the intestine absorbs solutes (Karasov and Hume, 1997).Pigeons are a good experimental model for studies of avian physiology in vivo. Some reasons for this are, (i) its cosmopolitan distribution, (ii) ease of capture and (iii) laboratory maintenance. Methodologically they are also a good subject be-cause they allow intestinal cannulation for dosage administration, as we describe here, as well as easy serial blood sampling for pharmacokinetics studies (Chediack et al., 2001).Our main objective was to adjust and improve a needle catheter duodenostomy technique, to study intestinal bioavailability of compounds by pharma-cokinetic techniques.MATERIAL AND METHODSAnimal care and housingAdult Feral Pigeons (Columba livia; Linneo, 1758) were captured with a live trap near the Universidad Nacional de San Luis Campus (San Luis, Argentina). The birds were housed individually in cages indoors under relatively constant environmental conditions (25.2 ± 0.3°C, relative humidity of 50 ± 9%) on a photoperiod of 12:12 hours (Light: Dark) with wa-ter and food ad libitum (normal food for breed-ing broilers, Cargill). We used a mix of a broad spectrum anthelminitc medication to treat internal parasites (Nevugon-Bayer 0.03% and Piperazine 1.5 g/l of water) and externally with Nevugon-Bayer 0.3%. Animals were acclimated to laboratory con-ditions prior to use in experiments. Animal care and trial protocols followed the guidelines of the Universidad Nacional de San Luis.Preparation of the cannulasCannulas were made with a nasogastric catheter (PVC/silicone composition), a common hypoal-lergenic material used in surgery and suitable for chronic use. The nasogastric catheter was cut at a length of 45 mm. The barrel of the cannula was 25 mm long, the outer and inner diameters were 2 and 1.4 mm, the extremity was rounded and a little support consisting of the same material was added. We added a support made of PVC/silicone to fix in the abdominal muscle, and we adapted an i.v. cath-eter (FEP polymer catheter 20G) inside the cannula to avoid obstruction by intestinal fluid (Figure 1).Surgical proceduresBefore cannulation surgery, the pigeons were ac-climated over 12–13 days in an animal room. The animals fasted 18 h before the cannulation surgery (enough time to empty the crop). Anesthesia was induced with ketamine (40 mg/kg) and xylacine (10 mg/kg) diluted in physiological saline solution and administered through an injection in the pec-toral muscle (Paul-Murphy and Fialkowski, 2001). The bird was placed in an operating table and the body fixed with thread. The operation zone was dis-Figure 1. Cannula used for intraduodenal cannulation. The nasogastric catheter was cut at a length of 45 mm. The barrel of the cannula was 25 mm long (1), the extremity was rounded and a little support consisting of the same material was added (2). We add an additional support (3) to fix in the abdominal muscle. Finally, we adapted an i.v. catheter inside the cannula (4)Veterinarni Medicina, 55, 2010 (8): 383–388 Original Paper385infected with chloroxylenol and the feathers were removed with vaseline. The abdomen was open longitudinally with a surgical knife, between the xiphisternum and the pubic bones. To access the intestinal cavity, a longitudinal incision was done (~ 3 cm), and the duodenum was exteriorized. The cannula (PVC/silicone catheter) was inserted in a small incision and fixed with thread to the intestine for one of the supports, and afterwards fixed to the abdominal muscle with another support (Figure 2). Finally, the skin was sewed.Birds were moved to a recovery room outside the surgery room, and observed for three to four hours after the surgery. The birds were fully awake six hours after surgery. Drinking water was offered immediately and food a day after surgery. The bioavailability ex-periments were performed one week after surgery.Material and reagentsCatheter K33 (Koler S-20. Argentina), FEP pol-ymer catheter 20G (Becton, Dickinson. Brazil). l-rhamnose (C6H12O5, MW = 164.2), ZnSO4, ace-tonitrile and derivatization reagents were purchased from Anedra S.A. (Buenos Aires, Argentina) and Sigma Chemical Co. (St. Louis, MO, USA). Ketamine (Holliday-scott S.A.) and xylacine (Köning S.A.).Pharmacokinetic method to assay bioavailabilityTo measure absorption we used a pharmacoki-netic technique which involves feeding (by intradu-odenal administration) and injecting probes and Figure 2. Steps of the surgical procedure. Abdominal incision between the xiphisternum and the pubic bones (A), the small intestine is exteriorized (B), the cannula is inserted and fixed to the intestine (C), and the duodenal cannula is in position to perform experiments (D)Original Paper Veterinarni Medicina, 55, 2010 (8): 383–388386then measuring those probes in the blood at various times post-gavage or post-injection (Chediack et al., 2001). Birds were placed in observation cages and iso-osmotic solutions of l-rhamnose were admin-istered intraduodenally after 18 h of fasting. At this time the animal doesn’t have any nutrients in the crop and/or stomach. The volume administrated was 3 ml and the administration was completed within one minute.Blood samples (120 µl) were collected from the wing veins with a heparinized capillary tube. Ten to twelve blood samples (1.2–1.5 ml total, which accounts for < 10% of total blood volume were col-lected (Stangel, 1986). The samples were centrifuged for 3 min at 10 000 rpm in a hematocrit centrifuge and plasma was separated and stored at 0°C for analysis. Plasma proteins were precipitated using 10% ZnSO4 and acetonitrile (Lam and Malikin, 1989). The rham-nose was derivatized for high performance liquid chromatography (HPLC) fluorescence detection by reductive amination with anthranilic acid (Du and Anumula, 1998). The HPLC system consisted of a Beckman automated binary system with a pump (model 126), an autosampler (model 507) and an interface (Model 406). Derivatives of carbohydrate probes in samples and standard solutions were de-tected with the Gilson 121 fluorescence Detector.ExperimentsExperiment 1. To test the efficiency of the can-nulation surgery procedure, we used ten pigeons weighing from 260g to 320g, five were cannulated and the other five were treated as controls (with-out cannulation). Each pigeon was housed in an individual cage following a random procedure. The body mass of each pigeon was registered in the morning (08:00–09:00 h) at different intervals of time (see Figure 3). Additionally, the surgery site of each pigeons was carefully examined and the correct operation of each cannula was tested.Experiment 2. To test the applicability of this model in the intestinal absorption research, four pigeons were cannulated and rhamnose (a passive absorption probe) bioavailability was measured following the pharmacokinetic method described above.Statistical analysisThe Shapiro-Wilk W-test was used to test for normality and the Levene Test was used to test for homogeneity of variances. Analysis of variance (ANOVA) was used to examine the effect on pi-geons with and without surgery. The significance level was set at P < 0.05.RESULTS AND DISCUSSIONThe body mass of pigeons was stable after two weeks of acclimation in the animal room (Figure 3). The cannulation surgery was performed at day 12–13 after arrival of the pigeons at the animal 2002503003504000 5 10 15 20 25 30Time (days)Body mass (g)Figure 3. Stability of body mass in pigeons in the animal room. Filled square and solid line: cannulated pigeons. Empty-triangle and dashed line: control, non-cannulated pigeonsVeterinarni Medicina, 55, 2010 (8): 383–388 Original Paper387room, and was successful in 100% of cases. Recovery from surgery was rapid and animals consumed water within a day, and food the next day. After the surgery, the body mass of cannulated pigeons continued to be stable until the end of experiments (Figure 3), and no significant differences could be detected between cannulated and non-cannulated animals (ANOVA P > 0.25). In the same way, Goring et al. (1986), found no adverse effects in the weight of pigeons fed by needle catheter duodenostomy versus control animals.Efficiency of cannulationWe tried several types of catheter with different flexibility: a 100% silicone catheter, FEP polymer catheter and a mix of silicone/PVC catheter. The most flexible cannula (silicone 100%) was better than rigid ones (FEP polymer and mix of PVC-silicone). A 100% silicone cannula stayed in the intestine for around two months, while the plastic catheter remained for about 3–4 weeks and a mix of silicone/PVC continued to work properly for at least one month. After this time the cannula was expelled by peristalsis without any harmful effect for the animal. Moreover, pigeons did not show any problem with regard to infection and cicatrisation. Three weeks after the expelling of the cannula, the condition of the intestinal and muscular tissue was normal, and we could make a new re-cannulation in the same site. Likewise, another study using a radiographic technique has reported that the duo-denum of a pigeon appeared normal four weeks after catheter removal (Goring et al., 1986).We decided to perform the two experiments us-ing a PVC-silicone catheter, because both of its stability over time its economic convenience.Application of the model in absorption experimentsThe technique developed for surgery and chronic studies has permitted us to obtain consistent and reproducible results in the same animal for differ-ent absorption experiments. We used the cannula for infusion of 3 ml of an iso-osmotic solution with l-rhamnose as an intestinal permeability marker. The administration was performed in < 1 min with-out anesthesia. Then, we took several blood sam-ples to analyze the absorption profile (Figure 4). The absorption profile of l-rhamnose determined in this study was in agreement with the results pre-viously reported by other authors in pigeons (Lavin et al., 2007).ImplicationsThe method described herein is simple, economi-cally attractive and useful for nutritional and eco-logical studies of the absorption of nutrients in the intestine in vivo. By using this cannulation method Figure 4. Absorption profile of L-rhamnose after intestinal administration of 3 ml of an iso-osmotic solution in can-nulated pigeons. The data points are means + S.E.M. (n = 4 pigeons)01002003004005006000 50 100 150 200 250 300 350 400Time (min)Rhamnose concentration (µg/g plasma g dose)   zOriginal Paper Veterinarni Medicina, 55, 2010 (8): 383–388388in combination with intraduodenal administration and intramuscular injection of compounds, it is possible to analyze the intestinal bioavailability in an in vivo model. In addition, it allows minimal use of anesthesia, which has previously been shown to be a problem in these kinds of studies (Uhing and Kimura, 1995).AcknowledgmentsThe authors thank Dr. Sergio Alvarez (Virginia Commonwealth University) for his thorough and critical reading of the manuscript. During the re-search Fabricio Damián Cid (FDC) held a post-doctoral fellowship from CONICET.REFERENCESBjarnason I, Macpherson A, Hollander D (1995): Intes-tinal permeability: an overview. Gastroenterology 108, 1566–1581.Caviedes-Vidal E (2003): Intestinal absorption and its implicances in ecology and evolution. In: Bozinovic F (ed.): Ecological and Evolutionary Physiology: Theory and Study Cases (in Spanish). Ediciones Universidad Católica de Chile, Santiago, Chile. 151–168.Chediack JG, Caviedes-Vidal E, Karasov WH, Pestchan-ker M (2001): Passive absorption of hydrophilic probes by house sparrows, Passer domesticus. Journal of Ex-perimental Biology 204, 723–731.Chediack JG, Caviedes-Vidal E, Fasulo V, Yamin J, Ka-rasov WH (2003): Intestine passive absorption of hy-drosoluble compounds by sparrows: effect of molecular size and luminal nutrients. Journal of Com-parative Physiology B 173, 187–197.Cid FD, Chediack JG, Caviedes-Vidal E (2005): Measure-ment of nicotine in blood of birds by reverse phase HPLC. Elimination of nicotine in pigeons. Preliminary data. Biocell 29, 42.Du P, Anumula KR (1998): Quantitative analysis of mon-osaccharides from glycoproteins by fast HPLC with Corresponding Author:Juan Gabriel Chediack, Dept. Biochemistry and Biological Sciences, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco 917, 5700 San Luis, ArgentinaTel. +54 2652 423789 int.129, Fax +54 2652 430224, E-mail: jg.chediack@gmail.comhighly sensitive fluorescence detection. Journal of Biomolecular Techniques 9, 31–33.Elia M, Behrnes R, Northrop C, Wright P, Neale G (1987): Evaluation of mannitol, lactulose, and 51Cr-labelled ethylenediaminetetra-acetate as markers of intestinal permeability in man. Clinical Science 73, 197–204.Goring RL, Goldman A, Kaufman KJ, Roberts C, Quesen-berry KE, Kollias GV (1986): Needle catheter duode-nostomy: a technique for duodenal alimentation of birds. Journal of American Veterinary Medical Asso-ciaton 189, 1017–1019.Karasov WH, Hume ID (1997): Vertebrate gastrointes-tinal system. In: Dantzler W (ed.): Handbook of Com-parative Physiology. American Physiological Society, Bethesda, MD. 409–480.Lam S, Malikin G (1989): An improved micro-scale protein precipitation procedure for HPLC assay of therapeu-tic drugs in serum. Journal of Liquid Chromatography 12, 1851–1872.Lavin SR, McWhorter TJ, Karasov WH (2007): Mecha-nistic bases for differences in passive absorption. Jour-nal of Experimental Biology 210, 2754–2764.Paul-Murphy J, Fialkowski J (2001): Injectable anesthe-sia and analgesia of birds. In: Gleed RD, Ludders JW (eds.): Recent Advances in Veterinary Anesthesia and Analgesia: Companion Animals. International Vet-erinary Information Service, Ithaca (www.ivis.org). Document No. A1409.0801.Perez M, Barber A, Ponz F (1993): In vivo increase of pas-sive intestinal absorption by cotransporters activation in rat. Revista Espanola de Fisiologia 49, 259–264.Roig T, Vinardell MP (1990): Intestinal perfusion in vivo for the study of absorptive processes. Journal of Com-parative and Biochemistry Physiology 98A, 3–7.Stangel PW (1986): Lack of effects from sampling blood from small birds. The Condor 88, 244–245.Uhing MR, Kimura RE (1995): The effect of surgical bowel manipulation and anesthesia on intestinal glu-cose absorption in rats. Journal of Clinical Investiga-tion 95, 2790–2798.Received: 2010–02–11Accepted after corrections: 2010–08–27

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Chronic cannulation in the small intestine of feral pigeons (Columba livia) to assess bioavailability

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Chronic cannulation in the small intestine of feral pigeons (Columba livia) to assess bioavailability

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