Research procedures

In the Stein Monogastric Nutrition Laboratory, some of the research procedures that are used include:

These procedures are all used on a routine basis in the laboratory and Dr. Stein and his graduate students have considerable expertise using the procedures to evaluate feed ingredients.

Apparent total tract digestibility and nutrient retention

For nutrients, such as minerals, that are not metabolized by microbes in the hind gut, apparent total tract digestibility (ATTD) values are usually measured. For energy and nutrients where absorption may occur in the hind gut, the ATTD are also measured.

In the Stein Monogastric Nutrition Laboratory at the University of Illinois, values for ATTD are measured by placing pigs in metabolism cages that allow for the total, but separate, collection of urine and fecal materials. The cages have an expanded metal floor that allows urine and fecal materials to fall through the floors as they are voided. A screen based floor under the metal floor will collect the fecal material, but the urine will flow into pans that are installed under the screen-floor.

There is a feeder installed in each cage and pigs are usually meal-fed. A daily amount of feed equivalent to 2.5 or 3 times the maintenance requirement for energy (i.e., 106 kcal ME per kg BW0.75) is offered in 2 equal meals. A screen is placed under the feeders to collect orts that are subsequently weighed. The quantity of orts is then subtracted from the total amount of feed offered to the animal for accurate estimation of the daily feed intake. Water is available at all times from nipple drinkers or bowl drinkers that are installed in the metabolism cages.

Pigs are usually fed the experimental diets in periods of 14 days. The initial 7 days of each experimental period is considered an adaptation period to the diet. In the morning meals on days 8 and 13, 0.5% ferric oxide, or another marker, is mixed into the diet. Fecal collections are initiated as the marker appears in the feces for the first time after day 8 and will cease when the marker appears in the feces for the first time after day 13. During the collection period, fecal materials are collected twice daily and stored at -20oC. Urine collections are initiated in the morning of day 8 and will cease in the morning of day 13. Urine is collected into urine buckets that are placed under the metabolism cages. The buckets are emptied morning and afternoon and 50 mL of 6 N sulphuric acid is added to the buckets every time they are emptied to prevent ammonia volatilization and microbial activity. The collected urine is weighed and a 20% sub-sample is collected and stored at -20oC. At the conclusion of the experiment, fecal and urine samples are thawed and mixed within animal and diet, and a sub-sample is collected for chemical analysis.

Following chemical analysis of diets, urine, and fecal samples, values for ATTD are calculated using Eq. 1:


ATTD (%) = [(Ni - Nf) / Ni] x 100 [1]


where ATTD is the apparent total tract digestibility of energy or a nutrient (%), Ni is the total intake of energy or a nutrient from day 8 to 13, and Nf is the total fecal output of energy or a nutrient originating from the feed that was consumed from d 8 to 13.

To estimate energy or nutrient balances, the retention of energy or nutrients are calculated for each pig using Eq. [2]:


Nr = [(Ni - (Nf + Nu)) / Ni] x 100 [2]


where Nr is the retention (%) of energy or a nutrient and Nu is the urinary output of energy or that nutrient from day 8 to 13 (g). Values for ATTD and retention of energy and fiber, Ca, P, Zn, and Cu are routinely measured in the Stein Monogastric Nutrition Laboratory at the University of Illinois. There are 40 metabolism cages for young pigs (from 5 to 25 kg) and 24 cages for older pigs (15 to 150 kg) available for this work.

Apparent ileal nutrient digestibility

Digestibility values for nutrients that may be metabolized in the hindgut of pigs are most correctly measured at the end of the ileum. Amino acids, starch, and ether extract are examples of nutrients for which accurate digestibility values cannot be measured using the approach for total tract digestibility. Therefore, apparent ileal digestibility (AID) values for these nutrients are measured to estimate the absorption from the intestinal tract of the pigs. The AID of nutrients is defined as the net disappearance of ingested dietary nutrients from the digestive tract proximal to the distal ileum. Values for AID are calculated from the flow and composition of digesta at the distal ileum of pigs and by relating the total ileal outflow of nutrients to the dietary intake according to Eq. [3]:


AID (%) = [(Nintake – Ileal Noutflow) / Nintake] x 100 [3]


where Nintake and ileal Noutflow represent dietary intake of a nutrient (g/kg DM) and the ileal outflow of that nutrient (g/kg DM), respectively.

To gain access to the ileal fluids, pigs are usually surgically fitted with a cannula. Several different cannulas may be used. In the Stein Monogastric Nutrition Laboratory, a T-shaped stainless-steel cannula is usually used. This type of cannula has been successfully inserted into the distal ileum of nursing pigs (from 3 to 5 kg), weanling pigs (from 6 to 10 kg), growing pigs (from 25 to 50 kg), finishing pigs (80 to 100 kg), and in gestating sows. Cannulas with different dimensions are used depending on the size of the pig. The cannula is surgically inserted 15 – 20 cm anterior to the ileo-cecal junction and extends through the body wall to the outside of the pig. A washer that is screwed onto the cannula barrel on the outside of the pig holds the cannula in place and a cap prevents ileal fluids from leaking out of the cannula.

Ileal cannulaFollowing the surgery, pigs are usually allowed to recuperate for 2 weeks before experimental diets are being fed. Pigs are housed in 1,2 x 1.5 m pens that are equipped with a metal triangle floor, a feeder, and a nipple drinker. During the experiment, pigs are usually fed each experimental diet for 7 days. The initial 5 days are considered an adaptation period to the diet, and ileal digesta are collected from the cannulas during 2 eight-hour periods on day 6 and 7. For collection of ileal fluids, the cap on the cannula is removed, and a 225 mL plastic bag is attached to the cannula barrel using an auto-locking cable tie. Bags are emptied as soon as they are filled with digesta, or at least once every 30 min. Digesta are stored at -20oC immediately after being collected to prevent microbial metabolism of the nutrients in the digesta.

Because the cannulas allow only for a portion of the ileal digesta outflow to be collected, an indigestible marker is included in the diet to calculated changes in nutrient concentrations prior to the distal ileum. At the University of Illinois, chromic oxide or titanium dioxide are usually used for this purpose. The marker concentrations in feed and digesta are then used to calculate AID according to equation [4]:


AID(%) = [1 - (Ndigesta / Ndiet) x (Mdiet / Mdigesta)] x 100 [4]


where Ndigesta and Ndiet represent the nutrient concentrations (g/kg) in digesta DM and feed DM, respectively, and Mdiet and Mdigesta represent the marker concentrations (g/kg) in diet DM and digesta DM, respectively.

At the University of Illinois, values for AID of amino acids, starch, and ether extract are routinely measured in pigs using the above approach.

Compartmentalization of intestinal nutrient absorption

Cannulated pigTo study the chemical, enzymatic, and microbial actions in different compartments of the intestinal tract, the tract can be compartmentalized using a multiple cannulation approach. By inserting intestinal cannulas in various places in the small intestine, the disappearance of nutrients in the GI tract may be compartmentalized. In a simple model, one cannula is surgically inserted into the duodenum and another cannula is inserted into the distal ileum. Pigs are fed experimental diets and samples of fecal materials, ileal digesta, and duodenal digesta are collected. An inert marker is included in the diet, and the apparent digestibility of nutrients is then calculated in each sample using equation [4] above. The disappearance of nutrients in the stomach, in the small intestine, and in the large intestine, respectively, may subsequently be estimated. If additional cannulas are inserted in the hindgut, digestibility values and microbial fermentation in different compartments of the large intestine may also be studied. In the Stein Monogastric Nutrition Laboratory at the University of Illinois, multiple cannulation of pigs has been used to study the digestibility of dry matter, nitrogen, and phosphorus in each section of the GI-tract. Likewise, the metabolism of exogenous enzymes and the breakdown of phytic acid in the digestive tract have also been studied in animals that were equipped with duodenal and ileal cannulas. Production of volatile fatty acids from pigs fed different sources of fiber was studied using pigs that were equipped with cannulas in the distal ileum and in the cecum.