pig model for studying nutr

Technical note: pig model for studying nutrient assimilation by the intestine and colon C. L. Kien, A. H. Ailabouni, R. D. Murray, P. A. Powers, R. E. McClead and J. Kepner J ANIM SCI 1997, 75:2161-2164.

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Technical Note: Pig Model for Studying Nutrient Assimilation by the Intestine and Colon1 C. Lawrence Kien2, Anton H. Ailabouni, Robert D. Murray, Priscilla A. Powers, Richard E. McClead, and Jon Kepner Department of Pediatrics, The Ohio State University College of Medicine and Children’s Hospital, Columbus 43205

ABSTRACT: We have developed a system for chronically catheterizing 10- to 25-d-old pigs that permits stable isotope tracer studies of intestinal or colonic assimilation of nutrients. This model also can be used to ensure constant enteral feeding or to assess the rate of entry into the terminal ileum of carbohydrates, fats, and amino acids. A plastic cannula with a luminal flange can be surgically placed in the stomach for tracer studies of sugar digestion or for controlled infusion of any formula diet. A similar cannula can be

placed in the cecum for infusion of tracer and(or) substrates for studies of fermentation. The cannula has been machined so that a washer and nut can be threaded onto it, allowing the entire apparatus to be fixed to the abdominal wall. The distal end protruding above the skin was tapered to fit standard i.v. extension tubing. A carotid arterial catheter was used to sample substrates for isotopic enrichment measurements.

Key Words: Pigs, Digestion, Fermentation J. Anim. Sci. 1997. 75:2161–2164

Introduction Studies of nutrient availability and metabolism in young pigs may require access to the vascular system and gut lumen (Walker et al., 1986). Moreover, young pigs are an excellent model for infant development, sugar digestion, nutrition, and gastrointestinal physiology (Pond and Houpt, 1978; Miller and Ullrey, 1987; NRC, 1988; Shulman et al., 1988). Experimental models involving chronic vascular access in pigs have been described previously (Shulman et al., 1988; Wykes et al., 1993; House et al., 1994). This paper principally describes a modification of a previously described apparatus for cannulating the intestinal tract of pigs (Walker et al., 1986). The chronically

1This study was supported by NIH Grant No. HD19773 and the Children’s Hospital Research Foundation. The authors acknowledge the assistance of Charles Maxwell of the University of Arkansas and Greg Morgan of Oklahoma State Univ., who provided the prototype cannula for us to modify and advice on the surgical procedure for inserting the catheter, Edward A. Liechty of Indiana University, who advised us on maintaining patency of the vascular catheters, Phil Bowers and Anne Lewis of the Children’s Hospital Vivarium, and Rita Porter who offered editorial assistance. 2To whom all correspondence and reprint requests should be addressed. Received November 13, 1996. Accepted April 25, 1997.

catheterized and cannulated pig model that we developed permits controlled feeding and facilitates tracer studies of intestinal and colonic assimilation of nutrients.

Experimental Procedures Source of Animals and Preoperative Care The use of young pigs in developing this model was approved by the Children’s Hospital Animal Care and Use Committee. Standard Yorkshire/Hampshire pigs (10 to 28 d old) were used. The pigs were transported from the swine farm to the laboratory 2 to 3 d before surgery (on d 10 of life). For 2 to 3 d before surgery, the animals were fed standard sow milk replacement formula (Litter Life MR Base, 10−07−9800, Merrick Foods, Union Center, WI) to accustom them to artificial nipple feeding.

Surgical Procedures Induction of anesthesia was attained with xylazine ( 4 mg/kg i.m.; Rompun, Bayer Corp., Shawnee Mission, KS) and a combination of tiletamine HCl and zolazepam HCl ( 6 mg/kg i.m.; Telazol, Fort Dodge Laboratories, Fort Dodge, IA). Anesthesia was then maintained with isoflurane. The pig’s trachea was

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intubated with an endotracheal tube through which ventilation and anesthesia were effected. The right carotid artery was catheterized for measuring isotopic enrichment during tracer studies. Via a second wound in the skin, the distal end of a polyvinyl chloride catheter (20 gauge) was tunneled to an exit site different from the entry site, and a heparin lock was inserted. This heparin lock consisted of a 20-gauge nail that was inserted into the heparin-filled catheter and caused the fluid to overflow and prevented an air space that would allow blood to backflow and cause a clot. Each day, the distal end of the catheter containing the nail was cut off before checking catheter patency and irrigating the catheter and(or) using it for blood collection. In a similar manner, an external jugular vein catheter was inserted as a back-up for blood collection. A modification of a chronic ileal cannula was then inserted (Walker et al, 1986). Using an electric surgical knife (Solid-State Electrosurgery Model 3000A, Neomed Inc., Boulder, CO), a 20-cm vertical incision was made in the abdomen approximately 2 to 3 cm left of the midline. The colon was identified and retracted, and the cecum was isolated. The rest of the bowel was eased back into the peritoneal cavity. An oval purse-string suture ( 3−0 silk) was placed at the location where the cannula was to enter. Then, using the electric surgical knife, a small incision was made in the cecum, the cannula was inserted, and the pursestring suture was tightened. An incision was then made on the left posterior side of the pig, and the cannula was inserted through this incision. External washers were threaded onto the cannula protruding from the skin and gently tightened against the skin (Figures 1 and 2). A syringe cap was then placed on the cannula. The incision in the abdominal wall was sutured in three layers (peritoneum, fascia, and muscle; subcutaneous tissue; and skin). We used continuous sutures for the first two layers and interrupted sutures in the skin ( 5−0 synthetic braid of a glycolic acid homopolymer; Dexon S, Sherwood, Davis, and Geck, St. Louis, MO). After surgery was completed, the pig was awakened and fitted with a vest that housed the catheters (custom made by Lomir Biomedical, Quebeck, P. Q., Canada). This vest can be attached to a commercial tether assembly described below (Lomir Biomedical).

Description of Cannula The acetyl copolymer cannula used for the stomach, cecum, or ileum was modified from one originally designed for sampling excreta from the ileum (Walker et al., 1986). We found that the originally described cannula was too large for infant pigs, it did not allow a suitable connection for substrate and tracer infusions, and it required one to insert a catheter through the distal opening on the skin surface, but the inserted catheter could not be held in place. Therefore, we

Figure 1. Diagrams of the cannulas used in the stomach, cecum, or ileum. The upper diagram depicts the cannula most appropriate for the stomach or cecum, and the lower diagram shows the cannula best suited for the small intestine (e.g., ileum). The thread dimensions are 6 × 1 mm. The tolerance for all dimensions is .1 mm.

redesigned this cannula, which was then constructed in the Medical Service Shop of The Ohio State University College of Medicine. Figure 1 depicts two versions of the device. The top diagram shows the cannula with the rounded flange that fits best in the stomach, cecum, or colon. The bottom figure shows the cannula that was designed for the ileum; it has a longitudinal flange. Figure 2 shows cannulas placed in the stomach and cecum that are protruding from the skin. We use only the longer cannula at the present time, but there will be some variation in the length of the cannula protruding from the skin depending on the size of the pig and the particular surgical technique in each pig. Besides its smaller diameter ( 1 mm i.d.), our version of the original device has two features that allow us to conduct isotope infusion studies. First, the distal end of the cannula protruding from the skin surface is sufficiently long to fit into the circular metal aperture of the vest (Figures 1 through 3). This aperture connects to a flexible metal tubing (Figure 3). This tubing serves as a tether, which in our studies was used to allow the animal to move freely within the confines of a small cage during blood sampling and isotope and(or) formula or substrate infusion (Figure 3). The tether can be fixed vial Allen


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Figure 2. Exit sites for the cecal and gastric cannulas. The shorter cannula on the left was placed in the stomach, and the longer cannula on the right was placed in the cecum. Currently, we use the longer cannula for all purposes.

screws to the aperture on the vest and a clamp that attaches to the cage (Figure 3). Second, the distal end of the cannula was machined to fit tightly as the inner (or male) connection for a standard i.v. extension tubing that passes through the tether (Figures 1 through 3). In our studies, this tubing was connected to a blunted 20-gauge needle over which was threaded approximately 2 cm of 20-gauge i.v. catheter tubing. The catheter tubing was then connected to a clamp that was fixed to the wall of the cage (No. 5, Figure 3). This clamp has needle connectors on both sides for connecting, respectively, to the tubing running through the tether (with needle connector and catheter tubing, etc.) and a similar set-up of 20-gauge catheter tubing, a blunted needle connected to a stopcock, and extension tubing running to the infusion pump (Nos. 6−8, Figure 3). Although Figure 3 depicts an infusion set-up for one cannula, we have designed a modification of the vest and tether apparatus that allows simultaneous infusion or fluid withdrawal using two cannulas. This modification is available commercially via special order from Lomir Biomedical, Inc.

Results and Discussion Pigs undergoing surgery have resumed normal feed intake within 48 h after surgery and have gained weight normally for periods of 7 to 19 d before euthanasia (McClead et al., 1990). After our initial

pig studies, we successfully cannulated the cecum, stomach, and(or) the ileum in five pigs. In each, we have also placed carotid arterial and jugular venous catheters. In each case, both vessels have remained patent and, during necropsy, we have observed no evidence of catheter infection. Similarly, each of the gastric and cecal cannulas has functioned well without leakage around the outside of the cannula. We have observed no evidence of infection around the tract to the skin for these cannulas in these pigs. We did observe in one pig intermittent fever associated with a small, superficial collection of purulent material at the site of the abdominal incision. When cultured, this material grew E. coli, suggesting that fecal contamination of the site may occur. A transparent adhesive film dressing (Opsite, Smith and Nephew, Largo, FL) is now being used postoperatively for 48 h to prevent fecal contamination of the incision, and pigs are now treated for 2 d after surgery with gentamicin.

Application of the Model in Tracer Experiments Depending on the experiments scheduled on a particular pig, the cannula can be placed in the cecum, ileum, or stomach. We have used the cannula for infusion of unlabeled lactose or glucose into the cecum accompanied by infusions of stable isotopes of acetate, propionate, butyrate, lactose or glucose. We also have used the ileal site for estimating the rate of appearance into the distal ileum of digested lactose. Polyethyleneglycol 4000 was added to the formula to


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young pig’s resilience in tolerating the surgery without prolonged ileus or mechanical intestinal obstruction and without significant infectious complications. From our experience with pediatric patients with leaking gastrostomy or jejunostomy studies, we were particularly surprised that the cannula was not associated with any leakage around it. However, in each of the pigs in which this approach has been used, necropsy has revealed that the cannula is tightly bound to the abdominal wall and skin by fibrous tissue. Thus, we would suggest that this device could be useful for studies of infant pigs requiring controlled enteral feeding. Recently, we published data regarding the fermentation of sugar in a study with young pigs using this surgical model (Kien et al., 1996).

Implications This article describes a surgical approach that allows nutrients, or tracers for nutrients, to be infused into the stomach, intestine, and(or) colon of young pigs. The same surgical preparation can be used to sample nutrients from the same areas of the gastrointestinal tract. The model described here could be useful for a wide variety of nutritional, metabolic, gastroenterologic, and pharmacologic studies in young pigs. Figure 3. Photograph of the infusion set-up showing (1) the cannula, (2) the vest with metal aperture through which the cannula fits; (3) Allen screw on the connection for the tether to the vest; (4) the flexible tether that can be affixed with Allen screws to the vest; and (5) the clamp for attaching the tether to the cage. Note the needle connectors for tubing passing through the tether (6) and for the tubing going to the pump (7). Note also that the tether attaches to the clamp via Allen screws. Number (8) shows the infusion pump.

correct for the fraction of total intake reaching the ileum. A cannula with a slightly greater diameter (2.5 mm i.d.) was used in this case. As noted above, Figure 1 (bottom diagram) shows the latest version of the ileal cannula that fits better into the lumen of the small intestine. Finally, we have used the gastric site for controlled feeding of formula over several days and for tracer studies of lactose digestion. The size and disposition of pigs prevents forced oral feeding and catheterization in the awake state. By inserting cannulas and catheters under general anesthesia and then allowing recovery from surgery, continuous infusion isotopic experiments can be conducted in the awake state. We are impressed by the

Literature Cited House, J. D., P. B. Pencharz, and R. O. Ball. 1994. Glutamine supplementation to total parenteral nutrition promotes extracellular fluid expansion in piglets. J. Nutr. 124:396−405. Kien, C. L., R. D. Murray, A. Ailabouni, P. Powers, J. Kepner, L. Powers, and H. Brunengraber. 1996. Stable isotope model for assessing production of short chain fatty acids from colonderived sugar: Application in pigs. J. Nutr. 127:3069−3076. McClead, R. E., M. E. Lentz, and R. Vieth. 1990. A Simple Technique to Feed Newborn Piglets. J. Pediatr. Gastroenterol. Nutr. 10:107−110. Miller, E. R., and D. E. Ullrey. 1987. The pig as a model for human nutrition. Annu. Rev. Nutr. 7:361−382. NRC. 1988. Nutrition of the Pig. National Academy Press, Washington DC. Pond, W. G., and K. A. Houpt. 1978. Nutrition. In: The Biology of the Pig. p 276. Comstock Publishing Associates, Ithaca NY. Shulman, R. J., S. J. Henning, and B. L. Nichols. 1988. The miniature pig as an animal model for the study of intestinal enzyme development. Pediatr. Res. 23:311−315. Walker, W. R., G. L. Morgan, and C. V. Maxwell. 1986. Ileal cannulation in baby pigs with a simple t-cannula. J. Anim. Sci. 62: 407−411. Wykes, L. J., R. O. Ball, and P. B. Pencharz. 1993. Development and validation of a total parenteral nutrition model in the neonatal piglet. J. Nutr. 123:1248−1259.


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