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Nutrition Research 27diets of animals (humans, canines, pigs, rodents) will lower1. Introduction

Increasing the viscosity of digesta in the gastrointestinal

tract through diet manipulation with dietary fiber has been

shown to significantly reduce postprandial glucose response

in humans, pigs, canines, and rodents, indicating a potential

mechanism for management of diabetes and abnormal

carbohydrate metabolism [1-4]. Although it has been

demonstrated that ingestion of viscous fibers added to the

postprandial blood glucose concentration and increase

viscosity of intestinal contents, there is no single standard-

ized method to measure viscosity of gastrointestinal tract

contents. Interpretation of digesta viscosity data is difficult

owing to lack of standardized methodology and presentation

of data.

In the case of fluid digesta from the stomach and small

intestine of monogastric animals, an evaluation of viscosity

is a complex and difficult task. The general equation toKeywords: Viscosity; Digesta; Mixer viscometry; Canine; Intestiinvestigation of the role of diet in viscosity.

D 2007 Elsevier Inc. All rights reserved.0271-5317/$ see fro

doi:10.1016/j.nutres.2

4 CorrespondingE-mail address: gBecause increasing viscosity of gastrointestinal tract contents has been shown to alter physiologic

responses in many species, and no single standardized method of intestinal viscosity measurement

exists, effects of diet and measurement techniques were studied in a canine model. Three

experiments were conducted to evaluate the use of mixer viscometry, effects of freezing,

centrifugation, time of sampling, and dilution of digesta, and effects of diet on viscosity of canine

ileal digesta and simulated small intestinal digesta viscosity. Digesta viscosity values measured at

218C were within 5% of those at 218C after a 24-hour freeze. Viscosity constants of whole digestasampled between 8:00 am and 2:00 pm daily ranged from 6575 to 32692 mPa d s and 7.47 to 9.03mPa d s after centrifugation. Digesta sampled between 2:00 pm and 8:00 pm daily had viscosityconstants of whole digesta ranging from 7137 to 17345 mPa d s and 6.42 and 9.46 mPa d s aftercentrifugation. Digesta samples diluted with Millipore filtered water, to determine whether a dilution

factor could be used to estimate viscosity, had drastically underestimated viscosity constants. Ileal

digesta viscosity constants were similar for dogs fed test diets varying in carbohydrate source and

ranged from 6901 to 12590 mPa d s. During simulated small intestinal simulation, viscosity peakedbetween 6 and 9 hours. Data indicate that alteration of digesta by centrifugation or dilution

underestimates viscosity data. Viscosity of whole digesta can be measured using mixer viscometry.

Variations in diet ingredients appear to alter intestinal digesta viscosity, indicating further need forAbstractDiet and measurem

small intestinal digest

Cheryl L. Dikeman, Kathleen A. Barry,Division of Nutritional Sciences and Department of Ani

Received 8 February 2006; revised 20 Dnt matter D 2007 Elsevier Inc. All rights reserved.

006.12.005

author. Tel.: +1 217 333 2361; fax: +1 217 333 7861.

cfahey@uiuc.edu (G.C. Fahey).t techniques affect

iscosity among dogs

chael R. Murphy, George C. Fahey Jr4ciences, University of Illinois, Urbana, IL 61801, USA

ber 2006; accepted 21 December 2006

(2007) 5665

www.elsevier.com/locate/nutrescalculate viscosity is shear stress divided by shear rate.

Shear rate is the velocity gradient established in a particularfluid due to an applied shear stress, such as a contraction in

the gastrointestinal tract of an animal [5]. Gastrointestinal

Approximately 30 mL of fresh ileal digesta was placed in

ition Rtract shear rates in animals have not been established and

may vary considerably with sampling location, individual

animal, meal composition, and gut motility. It can be

assumed that digesta is a slurry exhibiting non-Newtonian

shear-thinning behavior. According to this flow behavior, as

shear rate increases, viscosity decreases; therefore, multiple

shear rates should be used to measure viscosity. Many

researchers, however, have measured viscosity of digesta

from experimental animals at only 1 shear rate, making

comparisons among studies difficult [1,6-11]. Because a

vast range of shear rates was used, comparing the resultant

digesta viscosities is neither useful nor valid. Flow profiles

should be obtained for liquid digesta across various shear

rates rather than estimation of viscosity at only 1 shear

rate [12,13].

Another factor contributing to the difficulty in measuring

viscosity of digesta is concentration and size of undigested

particles in the sample. Particulate matter may interfere with

viscosity instrumentation and, therefore, many researchers

centrifuge and (or) dilute digesta samples with water before

measuring viscosity to remove large particles or dilute them.

Removal of particulate matter results in a more homogenous

sample [7,14-17]; however, this may remove particles that

contribute to viscosity. A solution to this problem may be

the use of mixer viscometry to measure the viscosity of

whole digesta. Mixer viscometry was developed to address

problems including large particle size and (or) particles

settling out of solution. Mixing involves intermingling of

2 or more dissimilar materials to obtain a desired degree of

uniformity. This uniformity is obtained by agitation to create

motion in the material, usually in the form of a rotational

viscometer with some form of mixing or vane spindles

[5,18]. Similar to other rotational methods such as cone and

plate geometries, mixer viscometry is limited by a limited

shear rate range.

To establish standard techniques to measure viscosity of

digesta collected from the gastrointestinal tract of animals, it

is necessary to understand the natural variation that exists

among individual animals and the factors that affect this

variation. An understanding of factors affecting viscosity

will be valuable in controlling animal variation in future

studies designed to measure viscosity of gastrointestinal con-

tents as well as in aiding in accurate viscosity measurement.

Although there is a great deal of literature available

discussing the effects of viscous fibers on the viscosity of

fluids such as gastric, small intestinal, and cecal contents

from animals, few data are available on the effects of

standard diets on digesta viscosity. The vast majority of

intestinal content viscosity has been measured after the

removal of solid particles through methods such as

centrifugation and dilution. A repeatable measurement of

viscosity on whole digesta would provide an excellent

opportunity to explore the effects of viscosity changes in the

gastrointestinal tract as affected by diet, ingredient(s), or diet

C.L. Dikeman et al. / Nutrmatrix, and allow for further investigations of links between

viscosity of gastric and intestinal contents and physiologic100-mL glass beakers with a diameter of 5 cm and allowed

to equilibrate to room temperature (238C). Although thistemperature is significantly lower than physiologic temper-

ature, it was necessary to avoid confusion between multiple

viscometer configurations where temperature control couldresponses such as digestibility, gut morphology, transit time,

blood glucose and cholesterol attenuation, and bowel health.

The objectives of this study were to determine the effects of

freezing, centrifugation, dilution, and various carbohydrate

sources in diets on intestinal viscosity of dogs.

2. Methods and materials

2.1. Experiment 1

2.1.1. Animals

Purpose-bred female dogs (n = 3; Butler Farms USA,

Clyde, NY) with hound bloodlines, an average initial body

weight of 27.9 kg (range, 27-28.9 kg), and an average age

of 3.7 years (range, 1.7-6.5 years) were used. Dogs had

previously been surgically prepared with an ileal cannula

according to Walker et al [19] with surgical and animal care

procedures approved by the University of Illinois Animal

Care and Use Committee. Dogs were housed individually in

kennels in a temperature-controlled room (218C) at theanimal care facility in the Edward R Madigan Laboratory,

University of Illinois. A 16-hour light/8-hour dark schedule

was used.

2.1.2. Procedures

Dogs were fed 400 g of the same commercial diet (Iams

Weight Control, The Iams Company, Dayton, OH) daily in

2 equal feedings of 200 g at 8:00 am and 8:00 pm. The

main ingredients of the diet were corn meal, chicken,

ground whole grain sorghum, and chicken by-product meal.

The dogs had been on this diet for several months before the

initiation of this experiment. Water was available ad libitum.

Ileal samples were collected for 1 hour between 10:00 am

and 11:00 am on 3 consecutive days. Ileal samples were

collected by attaching a sterile sampling bag (Fisher

Scientific, Pittsburgh, Pa) to the cannula barrel and around

the hose clamp with a rubber band. Before attachment of the

bag, the interior of the cannula was scraped clean with a

spatula and digesta discarded. During the collection of ileal

effluent, the dogs were encouraged to move about freely. To

deter the dogs from pulling the collection bag from the

cannula, Bite-Not collars (Bite-Not Products, San Francisco,

Calif) were used during collection times. After ileal effluent

collection, the cannula plug was put in place and the cannula

barrel and surrounding site were cleaned with a 10%

Betadine solution (Purdue Frederick Co, Stamford, Conn).

2.1.3. Viscosity

esearch 27 (2007) 5665 57not be used. All viscosity measures in the study were

determined at room temperature to alleviate any additional

ition Rvariation due to temperature. It would be assumed that

viscosity measured at physiologic temperature would be

lower than the viscosities presented in this study. In

addition, there are data to suggest that gas bubbles due to

continued fermentation at temperatures above 258C wouldlikely alter viscosity [20]. Ileal samples were mixed by hand

with a metal spatula for 60 seconds to homogenize the

sample. Viscosity was measured in duplicate with a Brook-

field LV-DV-II+ viscometer. An SSV-vane standard spindle

set (Brookfield Engineering, Middleboro, Mass) was used.

Spindle multiplier constants used were 2.62, 11.1, and 53.5

for spindles V-71, V-72, and V-73, respectively. These

constants were programmed into Wingather software

(Brookfield Engineering) for viscosity calculations. Viscos-

ity was calculated as a function of revolutions per minute

(rpm) over an rpm range of 0.3 to 10 (0.3, 0.6, 1, 2, 5, 10).

After initial measurement, samples were frozen at 208C.After 24 hours in the freezer, samples were removed and

allowed to equilibrate to 238C and viscosity measured aspreviously described.

2.2. Experiment 2

2.2.1. Animals

Purpose-bred female dogs (n = 6; Butler Farms USA)

with hound bloodlines, an average initial body weight of

26.1 kg (range, 19.4-28.2 kg), and an average age of

4.2 years (range, 3.0-7.8 years) were used. Dogs had

previously been surgically prepared with an ileal cannula

and were housed as previously described. Dogs were fed the

same commercial diet as was used in Experiment 1. The diet

met or exceeded the Association of American Feed Control

Officials [21] recommendations for dogs at weight mainte-

nance and had an ME concentration of 20628 kJ/kg. Dogs

were fed a total of 400 g of each diet divided equally

between 2 feedings occurring at 8:00 am and 8:00 pm. This

study was conducted over a 14-day period. Initially, a 6-day

diet adaptation phase preceded an 8-day collection of ileal

digesta. Ileal digesta was collected once daily during the

8-day collection period, and each sample collection lasted

1.5 hours in length. Sample times rotated by 1.5 hours each

day with the first-day collection beginning at 8:00 am.

Collected ileal digesta was immediately frozen at 208C.Before the viscosity measurement, ileal digesta was

composited for days 1 to 4 (8:00 am-2:00 pm) and days

5 to 8 (2:00 pm-8:00 pm) and thawed in a 558C water bathuntil samples reached room temperature (238C). Water wasoffered ad libitum by providing 1500 mL at 8:00 am and

8:00 pm daily into 2000-mL stainless steel bowls attached

to the cages. Before offering new water at 8:00 am and

8:00 pm, remaining water from the previous offering was

measured with a graduated cylinder and amounts recorded.

2.2.2. Viscosity

C.L. Dikeman et al. / Nutr58Viscosity was measured in duplicate as described in

Experiment 1. After the initial viscosity measurement, ilealdigesta samples were placed in 50-mL centrifuge tubes and

centrifuged at 11000 g for 10 minutes. Viscosity of thesupernatant (2 mL) was measured with a Brookfield LV-DV-

II+ viscometer with a Wells/Brookfield cone and plate using

a CP-41 cone and plate.

2.3. Experiment 3

2.3.1. Animals

Purpose-bred female dogs (n = 5; Butler Farms USA)

with hound bloodlines, an average initial body weight of

27.9 kg (range, 19.3-32.3 kg), and an average age of

3.2 years (range, 1.8-6.5 years) were used. Dogs had

previously been surgically prepared with an ileal cannula

and were housed as described previously.

2.3.2. Procedures

Dogs were fed 1 of 5 dietary treatments that consisted

of commercially available canine extruded diets. Data

have shown that various carbohydrates impact the

viscosity of intestinal fluid of many species including dogs

[2,4,6,8-10,13]. Therefore, the diets chosen differed in

carbohydrate ingredients and included either ground wheat

(GW) (Kibbles n Bits Original, DLM Foods L.L.C, San

Francisco, CA), brewers rice (BR) (Purina One, Nestle

Purina, St Louis, Mo), corn meal (CM) (Eukanuba Adult

Premium Performance Formula, The Iams Company), corn

meal + ground sorghum + ground wheat (Mixed) (Science

Diet Adult Original, Hills Pet Nutrition, Inc, Topeka, Ks),

or oatmeal (OatM) (Cycle Custom Fitness, DLM Foods,

LLC, San Francisco, Calif). Dogs were fed a total of 500 g

of each diet daily divided equally between 2 feedings

occurring at 8:00 am and 8:00 pm.

The experimental design was a 5 5 Latin squareconsisting of 10-day periods. A 6-day adaptation phase

preceded a 4-day collection of ileal digesta. Ileal digesta was

collected 3 times a day, with an interval of 4 hours between

collections. Ileal collections were 1 hour in length.

Sampling times on the remaining 3 days rotated 1 hour

from the previous days collection time. Ileal samples were

collected as described above. Ileal digesta collected at each

time was immediately frozen at 208C. At the end of eachperiod, ileal digesta was composited for each individual

dog. Composited ileal samples were allowed to thaw in a

558C water bath until they reached room temperature(238C). Samples were mixed by hand with a metal spatulafor 60 seconds. Approximately 30-mL duplicates of ileal

digesta were placed in glass beakers (5 cm in diameter).

Viscosity of ileal digesta was analyzed as described

above. If samples were too viscous to measure directly, they

were diluted 1:1 (w/w basis) with Millipore filtered water

(viscosity, 1 mPa d s).

2.3.3. In vitro digestion simulation

esearch 27 (2007) 5665For the in vitro digestion simulation, diets from

Experiment 3 were weighed (0.5 g) in duplicate and placed

10.3 8.6 1.7 20628

die

ition Rin 50-mL plastic centrifuge tubes. To simulate gastric

digestion, 0.2 N HCl (5 mL), 10% pepsin/HCl (w/v,

0.5 mL), and 0.1 mol/L (12.5 mL) phosphate buffer (pH 6)

were added to each tube. Solutions were adjusted to pH 2

with HCl (0.2 N) or NaOH (0.6 N). Tubes were stoppered

and incubated for 6 hours at 398C [22,23]. After the initial6-hour incubation, small intestinal simulation began with

the addition of 0.6 N NaOH (2.5 mL), 0.2 mol/L phosphate

buffer (pH 6.8, 5 mL), and 5% pancreatin solution (w/v,

0.5 mL), with adjustment to pH 6.8 with HCl (0.2 N) or

NaOH (0.6 N) [22,23]. Tubes were incubated at 398C for anadditional 18 hours. One set of substrates was removed from

incubation and frozen at 208C at 0, 3, 6, 9, 12, 15, and18 hours from initiation of small intestinal digestion

simulation. During the in vitro digestion simulation, vis-

cosity was measured using a Brookfield digital viscometer

(LV-DV-II+) with a Wells/Brookfield cone and plate

extension. Solutions were assayed using a CP-41 cone and

plate, and across rpm speeds of 0.3, 0.6, 1, 1.5, 2, and 3

(shear rates, 0.6, 1.0, 2, 3, 4, and 6 per second, respectively).

2.4. Chemical analyses

Diets used in all 4 experiments were analyzed for dry

matter (DM), organic matter (OM), and ash using the

Association of Official Analytical Chemists [24] methods.

Crude protein (CP) concentrations were calculated using

Leco nitrogen (N) values (N 6.25) for all samples usingAOAC [24] methods. Total lipid content was determined by

acid hydrolysis followed by ether extraction according to

the American Association of Cereal Chemists [25] and

Budde [26]. Total dietary fiber (TDF), soluble (SDF), and

Table 1

Chemical analyses of dietary treatmentsa

Diet DM OM CP AHF

%, Dry matter basis

Diet A 91.0 93.4 23.4 11.7

BR 93.3 92.8 28.2 19.4

CM 93.3 91.7 33.5 22.6

GW 91.1 91.6 24.9 11.9

Mixed 92.5 95.0 26.8 15.4

OatM 92.2 93.9 24.7 13.1

a Diet A used in Experiments 1 and 2; BR, CM, GW, Mixed, and OatM

C.L. Dikeman et al. / Nutrinsoluble dietary fiber (IDF) concentrations were deter-

mined according to Prosky et al [27]. Gross energy content

of the diets was determined by bomb calorimetry (Parr

Instrument Co, Moline, Ill).

2.5. Statistical analysis

Viscosity data were analyzed using NLREG software

(NLREG, Brentwood, Tenn) and SAS (SAS Institute,

Cary, NC). NLREG was used to develop a working

model of the viscosity flow curve data. Pseudoplastic

fluids can be represented adequately by the power law

equation ( y = a * xb) and termed power law fluids

[5,28-31]. In the equation, shear stress ( y) is a function ofthe consistency index or constant (a), shear rate (x), and a

dimensionless exponent (b) that indicates closeness to

Newtonian flow. The exponent will equal 1 for New-

tonian fluids and will be less than 1 for shear-thinning or

pseudoplastic fluids. The constant is a parameter propor-

tional to viscosity of power law fluids and is represented

in units of millipascals per second. Model development

allowed for the estimation of the constant and exponent

parameters in the above equation.

Viscosity data from Experiment 1 were analyzed for

NLREG parameters using NLREG software.

Nonlinear regression parameter data from Experiment 2

were analyzed using the Mixed models procedure of SAS

and included the fixed effect of treatment (whole or

centrifuged digesta) and the random effect of dog. Treat-

ment least squares means were compared using the

Bonferroni method to control experimentwise error rate.

A probability of P b .05 was accepted as statisticallysignificant. Data also were analyzed using the correlation

procedure of SAS to determine the linear relationships

between diet and water intake and viscosity parameters.

Data obtained from Experiment 3 were analyzed to

estimate nonlinear regression parameters using NLREG

software and SAS. The experimental design was a 5 5 Latin square. The statistical model included the fixed

effect of diet and the random effects of dog and period.

In vitro data did not meet criteria of normality tested by

the univariate procedure of SAS; therefore, data were log-

transformed before the statistical analysis. Data were

analyzed using the Mixed models procedure of SAS. The

experimental design was a factorial randomized complete

5.4 3.3 2.1 22190

8.5 5.9 2.6 22948

10.9 9.3 1.6 20105

6.7 5.1 1.6 21436

9.8 8.4 1.4 20704

ts used in Experiments 3 and for in vitro digestion simulation.TDF IDF SDF Gross energy, kJ/kg

esearch 27 (2007) 5665 59block design with diet serving as block. The statistical

model included the fixed effect of diet and the random effect

of replicate. Treatment least squares means were compared

using the Bonferroni method to control experimentwise

error rate. A probability of P b .05 was accepted asstatistically significant.

3. Results

3.1. Chemical analyses

Chemical analyses of diets tested in all 3 experiments are

presented in Table 1. Dry matter and OM concentrations

24-hour freeze from 3 dogs consuming a standard dieta

Frozen digesta

R2 Viscosity constant, mPa d s Exponent R2

0.99 13241 1.18 0.990.99 16084 1.31 0.990.99 15171 1.62 0.990.99 24878 1.41 0.990.99 17503 1.87 0.990.99 10846 1.50 0.990.99 12410 1.23 0.990.99 22270 1.00 0.990.99 16301 1.69 0.99

= a * xb), where y is shear stress, a is the viscosity constant, x is shear rate, and

exponent b1 for pseudoplastic fluids); R2 indicates the proportion of variation

ition Research 27 (2007) 5665were similar among diets and averaged 92.2% and 93.1%,

respectively. Crude protein and acid-hydrolyzed fat (AHF)

concentrations ranged from 23.4% to 33.5% and 11.7% to

22.6%, respectively. Total dietary fiber, SDF, and IDF

concentrations averaged 8.6%, 1.8%, and 6.8%, respective-

ly. The gross energy concentration of the 6 diets analyzed

ranged from 20105 to 22948 kJ/kg.

3.2. Experiment 1

Nonlinear regression viscosity constants, exponents, and

R2 values calculated for ileal digesta samples measured

fresh and after a 24-hour freeze/thaw are presented in

Table 2. Ileal digesta viscosity constants averaged 15052,

17116, and 16614 mPa d s for dogs 1, 2, and 3, res-pectively, over the 3-day testing period. After a 24-hour

freeze/thaw, viscosity values were within 5% of each other

with the exception of dog 2 day 2 and dog 3 day 2. A high

proportion of variation among digesta samples from dogs

over the 3-day sampling period was accounted for with

the nonlinear regression model based on high R2 values

(Table 2). All digesta solutions sampled during the 3-day

period exhibited non-Newtonian shear-thinning behavior

(decreasing viscosity with increasing shear rate) indicated

Table 2

Nonlinear regression viscosity parameters for ileal digesta fresh and after a

Dog Day Fresh digesta

Viscosity constant, mPa d s Exponent

1 1 13931 1.262 15563 1.303 15663 1.77

2 1 23726 1.442 16206 1.913 11416 1.52

3 1 12512 1.222 20813 0.833 16518 1.71

a Nonlinear viscosity parameters are based on the power law equation ( y

b is a dimensionless exponent indicating deviation from Newtonian flow (

explained by the nonlinear regression model.

C.L. Dikeman et al. / Nutr60by negative nonlinear regression exponent values ranging

from 0.83 to 1.91.3.3. Experiment 2

Dry matter intake (DMI), water intake, and nonlinear

regression viscosity constant averages for whole and centri-

fuged digesta samples are presented in Table 3. Nonlinear

regression viscosity constants calculated for whole digesta

ranged from a high of 22588 to a low of 7631 mPa d s.After centrifugation, viscosity constants were very low and

ranged from a high of 9.23 to a low of 6.96 mPa d s. Nosignificant linear correlation was detected between DMI,

water intake, and viscosity constants.

Nonlinear regression viscosity parameters for digesta

samples are presented in Table 4. When viscosity was

measured on whole digesta samples, nonlinear regression

constants ranged from 6575 to 32692 mPa d s duringmorning collections and 7137 to 17345 mPa d s duringafternoon collections. After centrifugation, nonlinear

regression viscosity constants were lower (P b .05) thanwhole samples and ranged from 7.47 to 9.03 mPa d sduring morning collections and 6.42 to 9.46 mPa d sduring afternoon collections. In addition, the nonlinear

regression model successfully explained 99% of the

variation among samples measured whole, indicated by

high R2 values; however, the model failed to successfully

explain the variation in 5 of 6 samples after centrifuga-

tion. Those samples that were not significantly modeled

using nonlinear regression were labeled with exponent

values of 1, indicating a lack of dependence of shear rate

on viscosity.

3.4. Experiment 3

Nonlinear regression viscosity parameters, DMI, and

ileal DM concentrations for dogs consuming diets varying

in carbohydrate ingredients are presented in Table 5. Ileal

DM concentration ranged from 11.7% to 14.6% and was

highest (P b .05) for dogs fed CM compared with dogs fedOatM and GW. No additional differences were detected.

During Experiment 3, 5 digesta samples were too viscousto measure using the Brookfield LV-DV-II+ viscometer

Table 3

Dry matter intake, water intake, and nonlinear regression viscosity

constants for whole and centrifuged ileal digestaa,b

Dog DMI, g/d Water intake, mL/d Viscosity constant, mPa d s

Whole Centrifuged

1 313 1066 15711 8.04

2 296 1022 8808 7.70

3 365 1095 22588 7.86

4 232 568 7631 7.64

5 361 902 12360 9.23

6 198 628 10104 6.96

a All nonlinear regression viscosity constants for whole digesta were

higher ( P b .05) compared with centrifuged digesta (pooled SEM1397.25).

b Correlations between DMI, water intake, and nonlinear viscosity

constants measured on whole and centrifuged digesta were not significan

( P N .05).;

,

t

Table 4

Nonlinear regression viscosity parameters for digesta measured whole and after centrifugation in morning and afternoon hours from 6 dogs consuming a

standard dieta,b,c,d

Dog Morning hours Afternoon hours

Whole digesta Centrifuged digesta Whole digesta Centrifuged digesta

1

Constant, mPa d s 14077 9.03 17345 7.05Exponent 1.58 1 1.20 0.61R2 0.99 NS 0.99 0.84

2

Constant, mPa d s 6575 7.47 11041 7.94

C.L. Dikeman et al. / Nutrition Research 27 (2007) 5665 61Exponent 1.38 1R2 0.99 NS

3

Constant, mPa d s 32692 8.67Exponent 1.27 0.96R2 0.99 0.98

4

Constant, mPa d s 8124 8.23Exponent 1.29 1R2 0.99 NS

5

Constant, mPa d s 12236 8.99Exponent 1.46 1R2 0.99 NS

6therefore, they were diluted with Millipore filtered water on

a 1:1 (w/w) basis. After measurement and application of the

appropriate dilution factor, nonlinear regression viscosity

constants were much lower than expected, ranging from

1585 to 5894 mPa d s (Table 6). These values should havebeen greater than the highest values calculated without

dilution (Napproximately 22,000) based on the maximummeasurement capacity of the viscometer. Three of the

diluted samples were obtained from the same dog consum-

ing the CM, Mixed, and OatM diets. Samples obtained from

one dog consuming the CM diet and one dog consuming

the Mixed diet also were diluted. Because of the drastic

Constant, mPa d s 10283 7.50Exponent 1.38 1R2 0.99 NS

a Nonlinear viscosity parameters are based on the power law equation ( y = a

b is a dimensionless exponent indicating deviation from Newtonian flow (expo

regression was not significant (NS; P N .05); R2 indicates the proportion of varib Constant mean estimates were used in the case of nonsignificant nonlinearc Ileal digesta sampled in the morning hours between 8:00 am and 2:00 pm and All nonlinear regression viscosity constants for whole digesta were higher

Table 5

Nonlinear regression viscosity parameters, apparent total tract dry matter and o

consuming diets containing various carbohydrate ingredients1,2

Item BR CM GW

Viscosity constant, mPa d s 12590 11291 9431Exponent 1.32 1.19 1.19Dry matter intake, g/d 358 353 303

Ileal DM, % 13.0a,b 14.6a 12.4b

a,b Least squares means in the same row that do not have common superscri1 Nonlinear viscosity parameters are based on the power law equation ( y = a

b is a dimensionless exponent indicating deviation from Newtonian flow (expon2 Diluted samples were not included in the statistical analysis.1.46 10.99 NS

12483 7.06

1.30 10.99 NS

7137 7.05

1.36 10.99 NS

12483 9.46

1.70 10.99 NSreduction in viscosity, those 5 samples were not included in

the statistical analysis.

3.5. In vitro digestion simulation

Nonlinear regression viscosity parameters for smallintestinal digestion simulations are presented in Table 7.

During small intestinal digestion simulation, the BR

treatment had a higher (P b .05) viscosity constant at6 hours compared with 9 and 15 hours.

Viscosity constants for solutions containing CM were

similar during the initial 6 hours of simulation and averaged

221 mPa d s; however, there was a reduction (P b .05) in

9925 6.42

1.30 10.99 NS

* xb), where y is shear stress, a is the viscosity constant, x is shear rate, and

nent b1 for pseudoplastic fluids); exponents were given as 1 if nonlinearation explained by the nonlinear regression model.

regression analysis.

d ileal digesta sampled in the afternoon hours between 2:00 pm and 8:00 pm.

( P b .05) compared with centrifuged digesta (pooled SEM, 1397.25).

rganic matter digestibilities, and ileal dry matter concentrations for dogs

Mixed OatM SEM P value

7934 10228 3134.7 NS

1.21 1.17 0.09 NS372 379 38.17 NS

12.7a,b 11.7b 0.59 b .05

pt letters differ ( P b .05).* x2), where y is shear stress, a is the viscosity constant, x is shear rate, and

ent b1 for pseudoplastic fluids).

viscosity beginning at 9 hours. Viscosity constants were

lower (P b .05) at 15 and 18 hours compared with the6-hour value.

Few differences were detected for the GW treatment. At

the initiation of digestion simulation, the viscosity constant

was lower (P b .05) than the viscosity constant after 6 hoursof digestion simulation. All other time point combinations

were similar. As was the case for the BR and CM

treatments, the GW treatment had the highest viscosity

constant value at 6 hours.

Viscosity constants for the Mixed treatment were lowest

(P b .05) at 6 and 15 hours of simulation compared with the9-hour value. No other differences were detected.

exhibit shear-thinning behavior [3,13,28-32]. Use of a

working model such as nonlinear regression provides

additional information about the viscosity/shear rate profile

not available if a single shear rate measurement is obtained.

This approach provides a more powerful tool for describing

viscosity characteristics in the gastrointestinal tract, even

though shear rates have not been established [13]. In the

current studies, nonlinear regression analysis indicated that

all solutions, with the exception of those that were

centrifuged, were non-Newtonian and exhibited shear-

thinning behavior as indicated by negative exponents.

Greater negative exponents are associated with a greater

dependence of viscosity on shear rate. It was expected that

ileal digesta collected from dogs would exhibit this type

of flow behavior because of the presence of particulate

matter within the fluid matrix. In support, Reppas et al [13]

reported that chyme collected from the duodenum and

jejunum of dogs exhibited shear-thinning or pseudoplastic

flow behavior.

Multiple methods of viscosity measurement have been

utilized in previous research to obtain a greater understand-

ing of this property in the gastrointestinal tract of animals.

Tube flow and rotational viscometers have been used to

determine flow properties, and effects of particle size on

Table 6

Nonlinear regression viscosity constants calculated for digesta samples of

dogs fed diets containing various carbohydrate ingredientsa

Dog BR CM GW Mixed OatM

1 21926 1585a 5362 9412 9094

2 9216 9349 5505 6835 8431

3 10331 3018a 12369 5894a 3026a

4 4715 6712 7713 7557 2943

5 16760 17813 16211 4811a 20446

a Indicates samples diluted 1:1 with Millipore filtered water.

h vari

7

7

d

5

9

C.L. Dikeman et al. / Nutrition Research 27 (2007) 5665624. Discussion

In the current studies, fluid viscosities have been

described using the power law equation to calculate a

viscosity consistency index or constant that is proportional

to viscosity. The power law equation describes solutions that

Table 7

Nonlinear regression viscosity parameters for solutions containing diets wit

Sample

0 3 6

BR

Constant, mPa d s 27d 47c,d 130c

Exponent 1.04 1.53 0.8R2 0.95 0.99 0.9

CM

Constant, mPa d s 254c,d 148c,d 262c,

Exponent 0.97 1.04 0.8R2 0.99 0.99 0.9

GW

Constant, mPa d s 14d 63c,d 77cExponent 1.50 0.92 0.78R2 0.99 0.99 0.99

Mixed

Constant, mPa d s 33c,d 14d,e 4e

Exponent 1.30 1.62 2.18R2 0.98 0.96 0.97

OatM

Constant, mPa d s 32 29 17Exponent 1.05 0.95 0.69R2 0.99 0.99 0.95

c, d, e Least squares means (5 diets, 7 time points; n = 35) in the same row that do1 Nonlinear viscosity parameters are based on the power law equation ( y = a

b is a dimensionless exponent indicating deviation from Newtonian flow (expon

explained by the nonlinear regression model.digesta collected from pigs, chickens, wallabies, and

brushtail possums [20,29,30,33-36]. Because of the effect

of particle size on viscosity, it seems relevant to use a

method that accounts for particles in fluid. Research has

been conducted on the application of mixer viscometry to

measure the viscosity of biologic materials [5,18].

ous carbohydrate ingredients during small intestinal digestion simulation1

Time, h

9 12 15 18

22d 36c,d 23d 75c,d

1.19 1.07 0.57 0.840.97 0.99 0.90 0.98

84c,d 95c,d 34e 43e

0.98 0.94 1.15 1.100.98 0.99 0.99 0.98

29c,d 34c,d 31c,d 48c,d

0.97 1.31 1.52 1.000.98 0.97 0.98 0.93

89c 31c,d 4e 17d,e

1.09 1.06 1.24 1.660.99 0.99 0.93 0.98

18 24 25 26

1.18 1.06 0.94 0.870.99 0.97 0.96 0.97

not have common superscript letters differ ( P b .05) (pooled SEM, 21.20).* xb), where y is shear stress, a is the viscosity constant, x is shear rate, and

2ent b1 for pseudoplastic fluids); R indicates the proportion of variation

ition RTo measure the viscosity of whole ileal digesta from dogs

without altering or removing particles or diluting the digesta

in any way, mixer viscometry was used. During the initial

experiment, on average, calculated viscosity constants

varied only by 12% among dogs. For each dog, there was

a greater variation among days. Furthermore, with the

exception of only 2 samples, viscosity values measured after

a 24-hour freeze and thaw to room temperature were within

5% of those measured on fresh digesta. Ability to freeze

digesta before measuring viscosity allows additional con-

venience when conducting experiments.

In Experiment 2, there was a 66% variation among dogs.

The larger variation in Experiment 2 may have been a result

of the sampling times. During Experiment 1, digesta was

sampled at the same time on each of the consecutive days.

In Experiment 2, digesta was sampled at rotating times that

changed every day during the collection phase.

During Experiment 2, there was no interaction or

correlation between DMI, water intake, and the calculated

viscosity constant. Water intake and diet intake were

measured but not controlled. Dogs were given a specified

amount of food and water but were not forced to consume

those amounts. Therefore, the variation in viscosity of ileal

digesta collected from dogs is not surprising owing to the

array of factors that might play a role in viscosity

measurement such as fluid intake, diet intake, fluid

secretions from the gastrointestinal tract, gut motility, meal

composition, particle size, and shear rate differences in the

gastrointestinal tract [5,30,31].

When ileal digesta samples from Experiment 2 were

centrifuged at 11000 g for 10 minutes, the viscosityconstants changed dramatically, resulting in very little

correlation between the 2 methods. In addition, according

to nonlinear regression analysis, 10 of 12 centrifuged

samples no longer exhibited non-Newtonian behavior based

on the exponent values of 1 and the nonsignificant model fit.

The removal of particles from digesta has been shown to

alter viscous characteristics of solutions, changing them

from non-Newtonian to Newtonian [34-36].

Researchers sometimes use centrifugation before mea-

surement of viscosity to control and remove large

particulate matter in samples. According to Tietyen et al

[37], centrifuging solutions containing oat bran and

hydrolyzed oat bran successfully separated the water-

soluble fraction from the large, insoluble particulate

fraction. This technique has been successfully used to

detect differences in viscosity of gastrointestinal contents of

rodents fed purified water-soluble fibers such as hydrox-

ypropylmethylcellulose and guar gum at concentrations of

40 to 50 g/kg of the diet [38-41]. When considering the

contents of the gastrointestinal tract, insoluble and large

particles contribute to viscosity; therefore, sampled digesta

should remain intact for viscosity measurement. Although

use of centrifugation might account for water-soluble

C.L. Dikeman et al. / Nutrfibers that contribute to viscosity in solutions [37-41], the

method removes the insoluble fibers and particulate matterthat also may contribute to viscosity characteristics in the

gastrointestinal tract [20,33-36].

Researchers have diluted extremely thick gastrointestinal

tract samples from experimental animals to produce a

digesta sample capable of being analyzed [6,42-44].

Because water has a viscosity of 1 mPa d s and is aNewtonian fluid, it was assumed that a dilution factor could

be applied to back calculate what the actual viscosity should

be. After dilution and nonlinear regression analysis, the

diluted samples had lower viscosity constants than their

undiluted counterparts. It was expected that those 5 solutions

would have much higher viscosity constants than the

highest values calculated for undiluted samples Unfortu-

nately, diluting the samples drastically underestimated the

viscosity constants in the present study, particularly for the

CM treatment. It might be expected that the CM treatment

would promote high viscosity in the gastrointestinal tract

owing to a relatively high concentration of SDF compared

with the other 4 diets. In addition, this diet contained higher

concentrations of CP and AHF compared with the other

diets, and these components also may contribute to viscous

characteristics. Lastly, ileal DM concentration was highest

for dogs consuming the CM diet. The higher concentration

of DM or solids in the ileal contents would be indicative of a

higher viscosity due to increased particulate matter in the

fluid and a lower water-to-solids ratio [5,30,34-36].

Cameron-Smith et al [8] reported that dilution of a

xanthan gumcontaining diet from an initial concentration

in solution of 18 to 12 g/kg resulted in no change in

viscosity. On the other hand, the same dilution of solutions

containing guar gum and methylcellulose resulted in

reductions in viscosity of 65% and 54%, respectively. These

findings, along with the substantial reductions in viscosity

found in the present study, call into question the validity of

using a dilution technique before viscosity measurement. In

addition, the drastic alteration in the viscosity constants after

dilution may support the idea that gastrointestinal tract

secretions play a very significant role in the viscosity of

gastrointestinal tract contents [8].

During small intestinal digestion simulation, dietary

proteins and carbohydrates were enzymatically digested.

The early phase of enzymatic digestion may contribute to

the ability of diet matrix structure to interact with fluid,

resulting in the increase in viscosities observed between

6 and 9 hours of digestion. As the enzymatic processes

continue, carbohydrate, protein, and fat structures in the diet

matrix would be modified and macronutrients prepared for

absorption and removal from the lumen of the small

intestine. This modification of structure likely causes the

reduction in viscosity observed with increased duration of

simulated digestion.

The drawback to in vitro investigations is the lack of

accounting for absorption of macronutrients and water, or

secretion of fluids into the gastrointestinal tract. It is still

esearch 27 (2007) 5665 63unclear how these processes and other factors such as diet

intake may impact gastrointestinal tract viscosity. Further

be avoided. These techniques of viscosity measurement will

ition Rassist researchers in understanding the impact that diet has

on characteristics in the gastrointestinal tracts of animals

including relevance to humans. Much research is still

needed to determine the effects of additional factors such

as fluid and diet intake, fluid secretions, gut motility, meal

composition, diurnal fluctuations, and shear rate differences

on viscosity of intestinal contents. Understanding the

viscous characteristics of animal intestinal contents will be

useful in defining the mixing, diffusion, and flow of

nutrients through the gastrointestinal tract.

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C.L. Dikeman et al. / Nutrition Research 27 (2007) 5665 65

Diet and measurement techniques affect small intestinal digesta viscosity among dogsIntroductionMethods and materialsExperiment 1AnimalsProceduresViscosity

Experiment 2AnimalsViscosity

Experiment 3AnimalsProceduresIn vitro digestion simulation

Chemical analysesStatistical analysis

ResultsChemical analysesExperiment 1Experiment 2Experiment 3In vitro digestion simulation

DiscussionReferences