Materials and Methods

Animals—Six healthy adult mixed-breed dogs of comparable body weight were included in the study. Dogs were 6 sexually intact females, 2 to 4 years old, with a mean ± SD body weight of 21.5 ± 1.8 kg. The dogs were owned by the Division of Laboratory Animal Medicine of the School of Veterinary Medicine at Louisiana State University. They were housed in outdoor runs at an Association for Assessment and Accreditation of Laboratory Animal Care International–accredited facility. Dogs were allowed exercise 3 times/wk. All dogs had been vaccinated against common diseases and had no known history or clinical signs of gastrointestinal tract disease. The study protocol was approved by an institutional animal care and use committee.

Evaluations prior to the experiments—Physical examination, a CBC, and serum biochemical analysis were performed on each dog. Only dogs with results within reference ranges for these assessments were included in the study. Body condition score was recorded for the dogs by use of a scale¹⁷ of 1 to 9 (1 = extremely thin, 5 = optimal, and 9 = extremely obese); all dogs had a body condition score between 4 and 6. Each dog was acclimated to a metabolic cage and gamma camera prior to onset of the study.

Diet—Dogs were allowed ad libitum access to a balanced dry kibble diet.^c The test meal for scintigraphy consisted of 225 mL of the kibble diet mixed with 1 jar (75 mL) of beef baby food^d and 5 mCi of ^99mTc-mebrofenin^e diluted in 5 mL of saline (0.9% NaCl) solution.⁸ The test meal contained 424 kcal, which provided approximately 30% of the daily estimated energy requirement of each dog, as determined on the basis of the following equation: 132 kcal/kg × BW^0.75, where BW is the body weight (in kilograms) of the dog.¹⁸ The kibble diet was placed in a plastic cup behind a radiation shield, and the ^99mTc-mebrofenin solution was poured over the kibble with constant mixing. The beef baby food was added to the meal 15 minutes later to increase palatability.

Experimental procedure—Food (but not water) was withheld from each dog for 18 hours. Each dog then received a 13 × 26-mm WMC^f PO, and the radio-labeled test meal was offered. Immediately after the meal was consumed (time 0), each dog was positioned in lateral recumbency over a large field-of-view gamma camera equipped with a parallel-hole collimator. Dogs were manually restrained without sedation for the duration of each of the image acquisitions by use of the gamma camera. A 60-second static image was acquired with the dog in both right and left lateral recumbency. Images were obtained by use of a 128 × 128-pixel matrix. Imaging sequences were repeated at 5, 15, 30, 45, and 60 minutes and then at 30-minute intervals for a maximum of 270 minutes. Because of concerns about radiation safety, the dogs were housed in metabolic cages after completion of the image acquisitions, and the radiation they emitted was measured with a Geiger counter until it was < 0.5 millirems at the surface of the skin over the abdomen (in general, approx 24 hours after ingestion of the radioactive meal).

Water was available ad libitum, and the dogs were offered the dry diet ad libitum as soon as the WMC had exited the stomach (indicated by an increase in pH of > 3 U on the data receiver display). Up to this point of each experiment, the WMC data receiver was kept within 3 to 5 feet of each dog at all times. When each dog was allowed to leave the metabolic cage and return to a run, it was fitted with a vest made of lightweight breathable fabric.^g The WMC data receiver was attached to the vest in the dorsal region of each dog and remained in place until the WMC was excreted in the feces. After excretion, the WMC was retrieved, deactivated, and thoroughly cleaned for another possible use. On the basis of the battery life estimated by the manufacturer, each WMC could be used twice.

The procedure was repeated 3 times in each dog at intervals of 1 to 2 weeks. The experimental procedure was repeated in each dog on the same day of the week.

Data analysis—Proprietary software^h was used to analyze the data retrieved from the data receiver. Tracings were obtained (Figure 1). The GET-WMC, SLBTT, and TTT were calculated. All gastric emptying and transit times were calculated on the basis of the pH data and compared with values recorded by the software. The SBTT was graphically defined as the interval between an increase in pH of > 3 U and a change in the pressure pattern from continuously high pressure (ie, MMC phase III) to isolated segmented contractions (ie, colonic motor complexes) associated with a decrease in pH that indicated passage through the ileocolic valve.^19,20 The LBTT was calculated as SLBTT minus SBTT.

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Graphic representation of pH (green), pressure (red), and temperature (blue) changes over time as a WMC moved through the gastrointestinal tract of a representative dog. Notice the abrupt increase in pH associated with intense motor activity (MMC phase III) when the capsule left the stomach. Passage through the ileocolic valve was associated with a decrease in pH (vertical line) and a change to a different motility pattern (colonic motor complexes). Exit from the body was characterized by a decrease in temperature. Pressure is reported in millimeters of mercury, time is reported in hours and minutes, and temperature is reported in degrees Celsius.

Citation: American Journal of Veterinary Research 71, 8; 10.2460/ajvr.71.8.903

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Scintigraphy—For each image obtained with the dog in right and left lateral recumbency, an ROI was manually drawn to include the entire stomach but to avoid adjacent bowel. Counts within these regions were then corrected on the basis of the decay for the physical half-life of ^99mTc from the image obtained at time 0. Geometric means of the decay-corrected counts within the left and right ROI (the square root of their product) were calculated, and time-versus-activity curves were generated. The straight portions of the curves between the beginning of rapid emptying and the beginning of slow emptying were then fitted by use of scientific graphing, data analysis, image processing, and programming software,ⁱ and the T_1/2-GES was calculated.

Statistical analysis—Data for analysis consisted of 3 paired values for GET-WMC and for T_1/2-GES for each of the 6 dogs. To evaluate the repeatability of each method, the CV was calculated for each dog; the mean CV for each method also was calculated. In addition, the ICCC was calculated for the GET-WMC and T_1/2-GES, and the obtained values were subsequently compared. An ICCC > 65% was considered evidence of moderate repeatability, and ICCC > 80% was considered good repeatability. Univariate analysis^j and an ANOVA^k were used for the statistical analysis.

To evaluate the relationship between GET-WMC and T_1/2-GES, the data were logarithmically transformed to achieve a normal distribution, and a nested ANCOVA then was performed. Variation of the GET-WMC was modeled on the variation in T_1/2-GES, which was nested within the random variance of dog. The percentage of the variance in GET-WMC attributable to dog and scintigraphy was calculated. We hypothesized that scintigraphy contributed no variation (and thus, GET-WMC and T_1/2-GES varied correspondingly) and that the dog contributed all the variation in GET-WMC. Univariate analysis^j and a nested procedure^l were used for the statistical analysis.

Results

All dogs tolerated both techniques well. Mean ± SD T_1/2-GES for each dog ranged from 99.9 ± 12.1 minutes to 181.2 ± 26.0 minutes and was highly repeatable among the 3 recordings within each dog (Figure 2). Mean ± SD GET-WMC for each dog ranged from 385.3 ± 9.3 minutes to 669.7 ± 88.3 minutes and also was highly repeatable among the 3 recordings within each dog (Figure 3).

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Results of the first (black bars), second (white bars), and third (gray bars) evaluation of GET by use of scintigraphy in each of 6 dogs. Each dog was evaluated 3 times at intervals of 1 to 2 weeks.

Citation: American Journal of Veterinary Research 71, 8; 10.2460/ajvr.71.8.903

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Results of the first (black bars), second (white bars), and third (gray bars) evaluation of GET by use of a WMC in each of 6 dogs. Each dog was evaluated 3 times at intervals of 1 to 2 weeks.

Citation: American Journal of Veterinary Research 71, 8; 10.2460/ajvr.71.8.903

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The CV for T_1/2-GES for each dog ranged from 6.8% to 15.5% (mean, 11.8%), and the CV for GET-WMC for each dog ranged from 0.3% to 10.7% (mean, 7.8%). The ICCC was 69% for T_1/2-GES and 71% for GET-WMC.

Results of the nested ANCOVA revealed that dog contributed a significant (P < 0.001) proportion of the variation for GET-WMC (82.1%), whereas scintigraphy did not contribute a significant proportion (17.9%). Thus, GET-WMC and scintigraphy varied correspondingly. This suggested that both methods provided similar results for evaluation of gastric emptying.

Values for SBTT, LBTT, SLBTT, and TTT were calculated from the WMC data. Mean ± SD SBTT for each dog ranged from 120.7 ± 31.1 minutes to 174.3 ± 7.8 minutes, whereas mean LBTT for each dog ranged from 900.3 ± 109.7 minutes to 2,498.0 ± 982.9 minutes, mean SLBTT for each dog ranged from 1,074.7 ± 102.4 minutes to 2,618.7 ± 954.8 minutes, and mean TTT for each dog ranged from 1,460.0 ± 93.3 minutes to 3,233.3 ± 953.4 minutes. The CV for SBTT for each dog ranged from 3.5% to 49.7% (mean, 16.7%), whereas the CV for LBTT for each dog ranged from 7.3% to 46.2% (mean, 23.4%), the CV for SLBTT for each dog ranged from 6.8% to 42.3% (mean, 21.3%), and the CV for TTT for each dog ranged from 3.8% to 32.4% (mean, 16.5%).

Discussion

The test meal was readily consumed by all dogs, and none were force-fed the meal. Because gastric emptying is influenced by a number of factors, volume and composition of the test meal were standardized and we attempted to minimize other variations. For example, all procedures for a specific dog were repeated on the same day of the week. Additionally, all dogs were housed under similar conditions and had the same physical activity. Dogs were acclimated to the procedures during a period of several days to minimize stress and obviate the use of sedation, both of which alter gastric emptying.^21,22,m

Few reports have provided information on intra-individual variability of gastric emptying in dogs. In 1 study,³ barium sulfate was mixed with ground kibble and fed to dogs (3 times in 9 dogs) to evaluate total GET (range, 7.5 to 14.3 hours; mean ± SE, 10.9 ± 0.76 hours). The SD for each dog in that study ranged from 0.5 to 2.1 hours. We calculated a mean CV of 10.0% for the data for that study,³ which is approximately the CV of 11.8% for T_1/2-GES in the study reported here. In another study,⁵ solid-phase gastric emptying was assessed 6 times in 4 dogs by use of the ¹³C-octanoic acid breath test. The CV for gastric emptying, time to peak ¹³CO₂ concentration, and half-dose recovery time were 16%, 14%, and 16%, respectively. In a third study,⁶ scintigraphy was performed 4 times in 4 dogs, and the mean for each dog of 240 to 378 minutes (group mean of 285 minutes with a CV of 22%) was obtained for 50% GET. In a fourth study,¹⁶ scintigraphy was performed and gastric emptying evaluated 3 times in 6 dogs by use of barium-impregnated polyethylene spheres. Mean ± SE for T_1/2-GES was 172 ± 17 minutes for the replication within dogs. It is difficult to compare these results because different test procedures that measured different endpoints were used in those studies. In humans, physiologic intraindividual variability of gastric emptying (expressed as CV) is described.^13–15 It reflects intrinsic biological variations.²³ Moreover, gastric emptying is a complex process that is controlled and influenced by various factors that might affect repeatability and cannot always be controlled or standardized. Evaluation of the use of scintigraphy revealed an intraindividual variability for gastric emptying that can vary between 7% and 21% for the solid component.^13,14,24,25 In the study reported here, intraindividual variability for gastric emptying in healthy dogs (T_1/2-GES = 11.8% and GET-WMC = 7.8%) compared favorably with results reported elsewhere. These variations must be considered when interpreting individual test results because they may make it difficult or impossible to detect pathological changes. However, intraindividual variations in humans are often negligible, when compared with values for changes in GETs associated with various motility disorders. Both scintigraphy and the WMC method allow differentiation between physiologically normal gastric emptying and gastroparesis in humans.^14,26 To our knowledge, no similar studies have been conducted in dogs.

After a meal has been broken up in the stomach, small particles will enter the duodenum, whereas large indigestible particles remain in the stomach and subsequently enter the duodenum during the interdigestive period via a burst of peristaltic contractions (phase III) as part of the MMC.²⁷ Because of its size, the nondigestible WMC given immediately prior to consumption of the radiolabeled meal leaves the stomach with the phase III MMC.²⁸ Therefore, GET-WMC reflects the time at which the entire solid meal has left the stomach. Expectedly, GET-WMC is in the same range as total GET evaluated by use of a contrast radiographic technique for a dry kibble test meal fed to mediumsized dogs.³

In contrast, T_1/2-GES represents the time at which the gastric radioactivity (expressed as the percentage of retained gastric activity at a given time) reaches < 50% of the immediate-postprandial radioactivity of 100%. The T_1/2-GES values reported in the present study were slightly lower than values reported for solid-phase T_1/2-GES in healthy dogs in other studies.^6,8,16,29,30 However, the amount and composition of test meals vary considerably and represent different proportions of the daily energy requirements for each dog. Moreover, differences in radionuclide markers, counting techniques, and methods of data analysis may explain the wide range of T_1/2-GES reported and render it difficult or impossible to make comparisons.

To evaluate the relationship between results for the WMC method and scintigraphy, the fact that each method measures a different endpoint had to be considered. Use of a nested ANCOVA revealed that changes in results for scintigraphy do not cause significant variations in GET-WMC and that any variations are simply attributable to differences within and among dogs. Thus, use of GET-WMC and scintigraphy provide similar results and are comparable for the evaluation of gastric emptying.

Values for SBTT, LBTT, and TTT measured by use of the WMC were larger than those reported elsewhere for healthy dogs.^6,31–34 However, in those studies,^6,31–34 TTT was assessed via different methods (small colored plastic beads or scintigraphy) in dogs with a wide range of body sizes or in dogs with a smaller body size. Large intraindividual variability was detected for the intestinal transit times in healthy dogs in the present study. This was more pronounced for LBTT (CV = 23.4%) than for SBTT (CV = 16.7%). Two of 6 dogs had a considerably larger SLBTT and TTT because of a larger LBTT during the first of the 3 repetitions. Without the values for those 2 dogs, mean CV for LBTT, SLBTT, and TTT would have only been 13.7%, 12.5%, and 10.0%, respectively. The only information available on LBTT in dogs is from a study³¹ in which investigators evaluated large intestinal transit of radiopaque markers in 10 healthy dogs. The mean residence time for polyethylene spheres (5 mm in diameter) in the colon ranged from 5 to 27 hours in that study.³¹ This underscores the profound physiologic variability of colonic transit in dogs. Considerable intraindividual variations in large intestinal transit times have also been reported in humans.^35,36 Despite identical diets, colonic transit is highly variable. This suggests an intrinsic variability in colonic function.^35,36

A lower CV was detected for WMC than for scintigraphy (7.8% vs 11.8%) in the study reported here. Although scintigraphy is considered the criterion-referenced standard for assessment of gastric emptying, it is not without variation.^6,16 The GET-WMC and T_1/2-GES varied in a similar manner, as determined by use of the nested ANCOVA. This was supported by calculation of the ICCC, which was moderate and equal for both methods. Thus, it indicates that repeatability of measurements obtained by use of the WMC method is as good as or superior to that of measurements obtained via scintigraphy. A limitation of scintigraphy was the user-dependent nature for the manually drawn ROIs at certain time points because of the overlap of activity in the small intestines or colon and the stomach, which was especially evident on the images obtained with the dogs in right lateral recumbency. This limitation has been reported by others.^8,37

The WMC method was suitable for use in evaluating gastrointestinal tract motility. The WMC can be used in dogs in their familiar home environment and does not require manual restraint or isolation of the dogs because of concerns about radiation safety. In this study, the vest and data receiver were tolerated well by the dogs. Additionally, there was no failure of the WMC, and every capsule was retrieved, inactivated, cleaned, and reused a second time. However, the size of the WMC does limit its use to dogs with a body weight of approximately ≥ 15 kg.

Analysis of results from the study reported here indicates that scintigraphy and the WMC method varied in a similar manner with regard to assessment of gastric emptying. There is moderate intraindividual variability with both methods, which must be considered when interpreting test results for a specific dog. Repeatability for WMC measurements is equivalent to that for scintigraphy, which is the current criterion-referenced standard used for the evaluation of gastric emptying. The WMC method is a nonradioactive technique, and the WMC can be easily administered and monitored on an outpatient basis for the assessment of gastric emptying in dogs.