Surgical Techniques Used in Amino Acid Nutrition Research


$$ q=({C_{\mathrm{ p}}}-{C_{\mathrm{ a}}})\times \mathrm{ PVBF}\times \mathrm{ d}t $$

where PVBF is portal vein blood flow (ml/min); C i is concentration of PAH infusion solution (mg/ml); IR is portal plasma PAH concentration (ml/min); PAHpv is portal plasma PAH concentration (mg/ml); PAHa is carotid arterial plasma PAH concentration (mg/ml); PCVpv is portal venous blood packed volume, PCVa is arterial blood packed volume; q is net absorption; C p is concentration of the nutrient in portal plasma; and C a is concentration of the nutrient in carotid arterial plasma (Huang et al. 2003).

16.2.1 Advantages of the Blood Catheter Method

The blood catheter method allows us to measure the net absorption of amino acids and their metabolites from the gastrointestinal tract.

16.2.2 Disadvantages of the Blood Catheter Method

The whole procedure is much complicated. It needs sophisticated methods and great technical expertise to do the surgery. Also, the expense of the whole surgery is much higher than other methods.

16.3 Slaughter Method

The slaughter method was first developed by Cori (1925). This procedure includes euthanasia of the pig through an approved method followed by removal of the ileum and subsequent collection of digesta (Yin et al. 2001; Tan et al. 2009).

The length of the small intestine removed may affect protein digestibility estimates. So the longer the section of intestine removed, the greater the chances that complete absorption has not occurred. After eating, feed takes about 6 h to reach the distal ileum in pigs (Donkoh et al. 1994). So the time point for digesta collecting is very important.

16.3.1 Advantages of the Slaughter Method


Although the slaughter method does have the advantage of causing minimal interference with the animal’s digestive tract prior to the time of sampling, it also allows us to take digesta from several parts of the digestive tract (Donkoh et al. 1994).



Any kind of diet can be used to determine amino acid digestibilities.



Experimental period is shorter and the procedure is relatively simple.


16.3.2 Disadvantage of the Slaughter Method


It is difficult to obtain representative digesta samples, because the optimal sampling time is easily influenced by diet type and other factors (Buraczewski et al. 1971; Rerat 1972). In addition, only one sample of ileal digesta can be obtained per animal. So this method needs more animals than other methods to obtain reliable results.



Furthermore, there may be shedding of mucosal cells into the gut lumen at death, which interferes with digesta nitrogen (N) content (Badawy et al. 1957, 1958; Fell 1961). Especially electrical current may cause cell sloughing in the gastrointestinal tract leading to contamination of the amino acid content of the digesta (Sauer and de Lange 1993; Albin et al. 1999).


16.4 Cannulation Technique

Due to the disadvantages of slaughter method, cannulation techniques were developed. Cannulation techniques consist of inserting a device (cannula) into the ileum to allow access to digesta in the gastrointestinal tract from the outside of the body directly and it has become a popular method in the determination of nutrient digestibility in pigs (Yin et al. 1993a, 2002).

16.4.1 Simple T-Cannulation

The Simple T-cannulation (STC) method was invented by Cunninghem et al. (1963) and later improved by Dierick et al. (1983). This procedure mainly includes insertion of a T-cannula into the terminal ileum. The T-cannula could be a full- or a half-round T-cannula. The full round T-cannula forces digesta to travel through the lumen of the cannula at all times, which can lead to increased blockage problems, especially with high fiber diets. In order to reduce the incidences of blockage of full-round T-cannula, the half-round T-cannula was developed (Sauer and de Lange 1993; Albin et al. 1999). The flange of the cannula is inserted into the ileal lumen and the barrel is exteriorized through the body wall, usually on the right side in a region bordered by the last rib, kidney, and the point of the hip (Sauer and de Lange 1993).

Advantage of the STC Technique

The STC method is widely used and has many beneficial characteristics, including the following:


It is a relatively quick and simple surgical procedure.



It little influences on the process of digestion when sampling.



It maintains a normal physiological state, which is advantageous for research involving the gastrointestinal tract (Sauer and de Lange 1992).



Digesta blockage and leakage risks remain low when compared with the FVRC technique, especially when half-round T-cannula is used.



It does not transect the small intestine wall, a practice that leaves the myoelectric complex intact (Sauer and de Lange 1992).


Disadvantages of the STC Technique


It remains difficult to quantitatively sample ileal digesta because of the unequal distribution of the liquid and solid phase of digesta and the possibility of blocking digesta when a full-round T-cannula is used (Graham and Aman 1986; Zebrowska 1978; Sauer and Ozimek 1986; Schroeder et al. 1989; Leterme et al. 1990). In addition, because of the position of the cannula, the digesta collected has not passed through the entire length of the small intestine.



In order to measure digestibility, an indigestible marker has to be used.



When high levels of fibrous dietary ingredients are used, blockage and leakage occur frequently so that the accuracy of results may not be optimal.


Cannula Design

Some points must be considered in order to design the cannula so as to improve its practical use.


In order to make room for more threads making the ring more stable, a thicker inner edge incorporated into the ring has to be designed.



Taking into account the animal care and protection, the ring must have a smooth outer edge in order to protect the skin.



Two holes can be placed in the ring to facilitate the use of a spanner wrench for ring adjustments, thus eliminating the need for notches on the outer edge of the ring.



Generally, a cannula barrel plug is made from nylon which can fit the cannula barrel very well. So the plug prevents digesta from entering and blocking the cannula barrel between collection periods.



The length and width of the cannula flange must be designed according to the pig age and body size to make sure it is suitable for placement in the distal ileum (Wubben et al. 2001; Walker et al. 1986).


16.4.2 Postvalvular T-Cecum Cannulation

The Postvalvular T-cecum Cannulation (PVTC) was developed by van Leeuwen et al. on the basis of the simple T-cannula method (van Leeuwen et al. 1988a, b). Later on, de Lange et al. (1989), Jørgensen et al. (1992), and Yin et al. (2000a) used these techniques to measure the digestibility of different nutrients and experienced some problems such as…. According to the limitations and problems, Jennifer et al. (2001) added some modifications to the PVTC technique with success to determine nutrient digestibility in pigs.

This technique involves placement of a full-round T-cannula at the ileo-cecal junction (Köhler et al. 1992a, b; Sauer and de Lange 1993; Albin et al. 1999). After removal of the cecum, the T-cannula is placed between the ileum and the large intestine, allowing digesta to flow from the ileum to the large intestine or out of the pig into a collection bag.

Advantage of the PVTC Technique

This technique has similar advantages to those given above for the STC technique. However, additional advantages include the following:


It can allow almost quantitative samples collection (Yin et al. 2000b).



In the PVTC technique, a larger diameter T-cannula can be used (Sauer and de Lange 1993; Albin et al. 1999).



This method reduces variation among animals and the number of animals required to complete an experiment (Wubben et al. 2001)


Disadvantage of the PVTC Technique


It includes a more complex surgical procedure and removal of the cecum (Radcliffe 1999) which represents an unphysiological situation.



As well as in the situation of ileo-rectal anastomosis (IRA), it remains unclear if the remaining intestine has similar function when a segment of the gastrointestinal tract is removed (Radcliffe 1999).


Surgical Procedures for the PVTC Technique

Surgery Preparation

Surgery is performed on healthy pigs that have been fasted for 12 h prior to surgery. Pigs are premedicated by intramuscular injection of 4 mg Azaperone (Stresnil®) and 0.05 mg Atropine sulfate per kg bodyweight (Van Leeuwen et al. 1991) or 1.5 ml of a telazol–ketamine–xylazine solution (Wubben et al. 2001). The surgical table is prepared with a heating pad covered by a clean towel to keep the animal warm and dry during surgery (Wubben et al. 2001; Walker et al. 1986). The pigs are placed in the left lateral recumbency and the right side of the animals is washed with a detergent solution to remove any external manure or dirt. The surgical area (a 30 × 30-cm area surrounding the last two ribs) is shaved by using an electric razor (Wubben et al. 2001, Walker et al. 1986).

After anesthesia, the pigs are left with inhalation of O2/NO in a ratio of 3:2, Halothane (Fluothane) (Van Leeuwen et al. 1991; Wubben et al. 2001). Wubben et al. (2001) has suggested to maintain the initial 5 % (v/v) halothane concentration for 20 min. The halothane concentration can then be reduced to 1.5–2 % to maintain general anesthesia for the remainder of the surgery. Wubben et al. (2001) suggested that to provide a snug fit, the mask should include a flexible rubber portion next to the pig’s snout. Of course, it is difficult to be completely sure that there is no anesthesia gases escaping into the surgery room, so it is necessary that safety procedures are put in place to protect surgeons from the anesthesia gases. Such procedures may include an exhaust fan with an attached exhaust tube placed near the pig’s head to vent the gas outside (Wubben et al. 2001). Pig’s breathing must be monitored during the entire surgery.

Obviously, all the surgical instruments, surgical drapes, and gowns must be sterilized by autoclaving to avoid infection during and after surgery. Cannulas are sterilized by immersion into a 2 % (v/v) solution of glutaraldehyde for at least 60 min.

Before cannulas are inserted into the ileum, they must be rinsed with sterile saline. The skin around the surgical area on the animal must be washed with 70 % ethanol and iodine. Sterile drapes are placed over the surgical area and the front and back legs (Wubben et al. 2001).

Surgical Procedures

Lidocaine with noradrenaline are administered in the area of the incision subcutaneously and intramuscularly for local anesthesia (Van Leeuwen et al. 1991). The detail surgical procedure can be done according to the procedures described by Wubben et al. (2001).

16.4.3 Steered Ileo-Cecal Valve Cannulation

A more recent technique for total collection of ileal digesta is the ileo-colic post-valve procedure which was developed by Darcy et al. (1980). This method maintains the integrity of the ileum and preserves the functional role of the ileo-cecal sphincter. Later Mroz et al. (1996) implemented a new cannulation technique called steered ileo-cecal valve cannulation (SICV). This method allows for a quantitative collection of ileal digesta.

The SICV cannula comprises of five parts: an inner cannula barrel, an outer cannula barrel, an internal ring attached to a nylon cord, an external ring, and a cylindrical stopper (Radcliffe et al. 2005). The cecum is not cut off when using the SICV cannulation method. The SICV cannula can be moved by using two rings; the inner ring is for the ileum and its distance to the ileo-cecal valve must be about 10 cm, depending on the age of the pigs. To let inner ring stay at the end of ileum, the external ring is made much smaller than inner ring. The external ring is at the end of ileum and attaches to the wall of cecum tightly. The T-cannula is fixed outside of the cut which is about 2–3 cm in cecum. At the time of sampling, the ileo-cecal valve will get into the T-cannula by pulling the nylon cord, meanwhile the external ring will block the space between the colon and the cecum. Mroz et al. (1991) compared the effect of the SICV and PVTC methods on growth performance and digestibility and found that the growth performance of SICV group is better, but the apparent digestibility of DM, organic matter and chloride is lower. The reason is likely that PVTC cannot get total digesta or that flow rate of digesta is different. Mroz et al. (1991) propose to use the SICV method only for 6–8 weeks after surgery because fibrous tissue between two rings provokes hyperplasia 13–14 weeks after surgery. Later Zhang et al. (2004) compared the method of SICV-cannula and T-cannula and found that ileal digesta samples from SICV-cannula are more homogenous than those from the T-cannula.

Advantages of the SICV Method


Sample collection is easy.



This method can maintain the integrity of the ileum and preserves the functional role of the ileo-cecal sphincter.



This method allows for a quantitative collection of ileal digesta.


Disadvantages of the SICV Method


SICV needs complicated surgery procedure and more labor and time cost than other methods.



SICV could only be used for 6–7 weeks after surgery, because of the fibrous tissue that develops between two rings due to hyperplasia 13–14 weeks after surgery (Mroz et al. 1991).


16.4.4 The Re-entrant Cannulation Technique

The re-entrant cannulation technique is also one of those that allow for total ileal digesta sample collection. This method consists of fitting two T-cannulas at two different sites of intestine and connecting the two cannulas using a bridge cannula. When getting samples, the bridge cannula is removed. Then, total digesta can be recovered from the proximal cannula. The digesta after analysis is warmed and put back into intestine through the distal cannula which allows for a situation that is close to the normal physiology and metabolism in pigs (Yin et al. 1991).

Depending on to the position of the cannulas, the re-entrant cannulation technique can be divided into two fore-valve re-entrant cannulation (FVRC) and post-ileo-colic valve cannulation (PICV).

Fore-Valve Re-entrant Cannulation Technique

The FVRC is when the proximal cannulation is made before the ileo-cecal valve. The FVRC can be divided into IIRC, ICRC, IORC, and IRRC. The detailed operation procedure can be found in previous studies (Cunninghem et al. 1962; Easter and Tanksley 1973; Laplace et al. 1985). van Leeuwen et al. (1988a, b) who designed the ileo-cecal re-entrant cannula for piglets have described in detail the technique used.

Advantages of the Re-entrant Cannulation Technique


Re-entrant cannulation is easy to operate.



The accuracy of data obtained by using re-entrant cannulation is very good.



When using the re-entrant cannulation technique, there is no need for a digestibility marker, thus avoiding the challenges associated with maker analysis and recovery.


Disadvantages of the FVRC Technique


Because the small intestine is completely cut off, the ileo-cecal valve does not work, which affects the normal excitation–contraction coupling which control the normal digesta flow in the small intestine.



The bridge cannula can easily drop off, with resultant problems of blockage and leakage, especially with diets containing high levels of non-starch polysaccharides.


Post-ileo-colic Valve Cannulation

Darcy et al. (1980) developed the ileo-colic post-valve cannulation technique. PICVC is when the proximal cannula is placed after the ileo-cecal valve. The technique involves the transsection of the intestine and the collection of digesta after the ileo-cecal valve. Although two cannulas are used in this technique, the re-entrant flow of digesta into the colon is not spontaneous. Also the surgery involved in this technique is rather difficult (Darcy et al. 1980). PICVC keeps the integrity of whole ileum, including the ileo-colic valve. However, the cecum is almost cut off totally and the proximal cannula is put into the residential cecum which is behind ileo-colic valve.

Advantages of PICVC


PICVC keeps the integrity of the whole ileum.



The result accuracy is higher than FVRC.


Apr 21, 2017 | Posted by in GENERAL | Comments Off on Surgical Techniques Used in Amino Acid Nutrition Research

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