Translational and Regulatory Sciences
Online ISSN : 2434-4974
Experimental Animal
Innovations in noninvasive methods for measuring gastrointestinal motility in mice
Kazuhisa KISHINoriyuki KAJIMasatoshi HORI
Author information

2021 Volume 3 Issue 1 Pages 22-29


In vivo assessment of murine gastrointestinal (GI) motility is useful for understanding GI diseases and developing effective therapies. The establishment of noninvasive measurement methods for mice will contribute to translational research bridging basic research and clinical practice, which can be a beneficial for maintaining quality of life in humans and animals. Recent advances in noninvasive diagnostic techniques have led to this update on the application and performance of available tests in mice. In vivo imaging techniques have been developed as noninvasive methods for the assessment of murine GI motility, and many of these methods have been applied to humans. Imaging techniques, including scintigraphy and ultrasonography, are frequently used in clinical practice. Basic data obtained using methods commonly used in clinical practice may be directly translated to clinical practice and are more attractive than those obtained using invasive methods. In this review, we provide recommended methods for noninvasively investigating gastric, small intestinal, and colonic motility in mice and detail the benefits of each test.


The development of noninvasive methods for mice will contribute to the understanding of gastrointestinal physiology in vivo and to the ethics of animal research. In particular, imaging techniques, such as diagnostic imaging, are advancing rapidly in clinical practice, and the idea of reverse-translating these into new noninvasive techniques for mice will be of great benefit in translational research. Noninvasive measurement techniques applied to humans and mice will bridge the physiological gap between basic and clinical data and improve reproducibility in the clinic.


Mice are the most commonly used laboratory animals in basic medical research. The advantage of using mice for research is that there are many established disease models, and a wide range of basic information is available [1]. Genetically modified mice are readily available, and mouse models provide insights into the mechanisms underlying many diseases. Additionally, mice offer an advantage in terms of breeding costs and have been widely used in experiments to predict biological responses.

In vivo methods for assessing murine gastrointestinal (GI) motility provide fundamental data for understanding the physiology of the GI tract, which is tightly regulated in vivo. Noninvasive tests are more attractive than invasive methods because they are often applied as diagnostic techniques in human and veterinary clinical practice. In fact, several tests used as clinical diagnostic techniques have been applied to mice, and the data fully reflect clinical data [2,3,4]. Thus, noninvasive measurement techniques in mice can be of great benefit in translational research, bridging basic research and clinical practice. Advances in gastroenterology require updated information on the application and performance of currently available noninvasive tests for the assessment of murine GI motility.

Noninvasive methods for measuring GI motility in mice have several advantages. First, noninvasive methods provide data for analysis over time, allowing for the tracking of disease progression and drug-induced changes in GI motility. Second, the method applied as a diagnostic technique in the clinic facilitates the extrapolation of objective data from preclinical studies to clinical practice. Since these data were obtained using common measurement techniques in basic research and clinical practice, they are expected to be directly applicable to clinical practice in humans and animals. Additionally, noninvasive measurement techniques that replace invasive methods will contribute to the principle of 3Rs (replacement, reduction, and refinement), a fundamental tenet of animal testing. In recent years, it has been estimated that more than 100 million mice and rats are used in animal experiments annually in the United States [5], raising concerns about the number of laboratory animals and appropriateness of welfare protection. The application of noninvasive measurement methods is essential to emphasize the principle of 3Rs and reduce the number of animals used in experiments.

The aim of this review was to provide concise information on noninvasive methods for measuring GI motility in mice. Table 1 lists the methods established to date for the noninvasive assessment of murine GI motility. For brevity, tests to assess nausea and vomiting associated with GI motility disorders are omitted here. Mice do not have a vomiting reflex, and there are few methods to objectively assess nausea and vomiting in mice. Currently, the method based on the ingestion of kaolin (pica behavior) is a noninvasive tool for assessing nausea and vomiting in mice [6, 7]. In the next section, we discuss both available tests and new tests that have been developed in recent years.

Table 1. Noninvasive tests of murine gastrointestinal motility available or in development
Gastrointestinal motor functions Tests Recommended approaches and future expectations
Whole-gastrointestinal transit Nonabsorbable marker · The most common approach is to monitor the timing of excretion of nonabsorbable markers such as charcoal, carmine red, and Evans blue in the stool.· Automated monitoring of defecation may provide a useful method for accurately measuring whole-gastrointestinal transit time in mice.
Fluorescence imaging
Detection of pellet production
Gastric motility Barostat · Recommended approaches for the evaluation of murine gastric emptying are scintigraphy and the breath test, as in humans. · Scintigraphy has the advantage of visually and quantitatively detecting changes in gastric emptying. · The breath test is a safe method that does not involve radiation exposure and is the most suitable method for longitudinal analysis with repeated measurements in the same animal.
Breath test
Fluorescence imaging
Small intestinal motility Fluoroscopy* · Imaging techniques are useful for visualizing peristalsis and segmental movements of the small intestine. · In vivo imaging modalities used in both humans and animals, such as ultrasonography, are expected to contribute to translational research.
Fluorescence imaging
Colonic motility Bead expulsion assay · A bead expulsion assay is a major tool for assessing murine colonic transit and serves as a model for in vivo studies to understand the physiology of colonic motility. · The advantage of ultrasonography is that dynamic and morphological assessment of colonic motility can be performed simultaneously in real time, in addition to the assessment of stool characteristics.
Detection of pellet production

MSOT, Multispectral optoacoustic tomography. *These imaging tests involve radiation exposure.

Whole Gastrointestinal Motility

Whole GI motility is assessed using GI transit studies. Oral administration of wireless motility capsules (WMC) is a useful test for whole GI transit in humans [8]. WMC technology can noninvasively determine gastric emptying, small intestinal transit, and colonic transit by using distinct changes in pH and pressure as landmarks.

On the other hand, the size of the WMC is a major limitation for its use in mice, and to the best of our knowledge, the WMC technique has not been applied for the evaluation of murine GI transit. Currently, the most common method for the assessment of whole murine GI transit is terminal experiments in which the timing of the first appearance of non-absorbable markers, such as charcoal [9], carmine red [10,11,12,13,14,15], and Evans blue [16,17,18,19], in the stool is measured. Several recent studies have shown that fluorescence imaging with indocyanine green [20] and diagnostic imaging [21, 22] are useful for measuring intestinal transit in non-terminal experiments. While single-photon emission computed tomography (SPECT) imaging has been used in many animal models, scintigraphy may be a promising method leading to better preclinical studies; however, scintigraphy involves prolonged restraint and the risk of radiation exposure, as well as high costs and the need for special facilities. Considering the restraint time and stress, the appropriate use of scintigraphy in mice may be limited to partial assessments such as gastric emptying.

In recent years, simple and efficient methods have been proposed that focus on reducing the stress that the experimenter places animals under. Several recent studies have shown that noninvasive methods that allow automated monitoring of feces allow for the accurate measurement of whole GI transit time in mice [23, 24]. Automated monitoring is made possible by using a camera to capture and detect feces labeled with non-toxic dyes over time. This technology has the advantage of being an easily achievable and labor-saving method. The presence of an experimenter stresses the animal and affects physiological responses and GI transit [25, 26]. The development of noninvasive and simple methods, such as automated animal monitoring, may reduce animal stress and improve the reproducibility of GI studies.

Gastric Accommodation

Gastric accommodation and adaptive relaxation in the gastric fundus are physiologically important mechanisms that result in increased gastric compliance and storage of ingested food [27]. Currently, measurement techniques for assessing proximal gastric function in humans include the barostat balloon test [28] and the drink test [29]. In the barostat balloon measurement, a balloon is placed in the gastric fundus, and changes in volume and internal pressure are recorded. The drink test is less invasive than the barostat balloon test and has the advantage of being inexpensive and easy to perform; however, the results of the drink test are not suitable for use in animals, including mice, because they depend on qualitative evaluation based on the subject’s senses. Therefore, the function of the gastric fundus in mice is assessed using barostat balloon measurements [30]. Whether a miniaturized barostat for mice has high maneuverability and the ability to detect gastric adaptive relaxation requires further validation.

In general, measurements to assess gastric accommodation are technically challenging, and the development of new modalities for the assessment of gastric accommodation remains controversial in both basic medicine and clinical practice. Several studies have shown that ultrasonography is a promising alternative method for the noninvasive assessment of gastric accommodation in humans [31, 32]. Imaging techniques such as ultrasonography may be applied to animals, including mice, as a noninvasive measurement technique in the future.

Gastric Emptying

Tests of gastric emptying are important for assessing the cause of gastroparesis, postprandial abdominal bloating, and vomiting with no apparent organic abnormalities. Several tests have been conducted to measure gastric emptying in mice. Useful noninvasive methods recommended for evaluating gastric emptying in mice include scintigraphy and breath tests, which indirectly detect the movement of contents using markers that are absorbed and metabolized in the duodenum and expelled in the breath. The interpretation of the results of these tests depends on the complete ingestion of the test meal and adequate observation time.

Scintigraphy to evaluate gastric emptying is the gold standard in humans and has been validated for use in mice [33]. The radionuclide used in this test is usually 99mTc, and the test meal for mice is typically made by mixing 99mTc-labeled albumin colloid or diethylenetriamine pentaacetic acid (DTPA) in a methylcellulose solution. This method has been used to investigate the pharmacological regulation of gastric emptying [34] and the effects of inflammatory conditions on gastric emptying and its treatment [35, 36]. Although scintigraphy involves high usage costs and radiation exposure, the benefits of visual and quantitative detection of changes in gastric emptying are attractive.

The breath test is a noninvasive method for evaluating the function of gastric emptying over time and quantitatively without radiation exposure. The breath test data correlate well with the results obtained from scintigraphy [37]; therefore, the breath test is frequently applied in mice. Stable isotopes of carbon-13 (13C) are used as detection markers, and 13C-octanoic acid and 13C-acetic acid are mainly used as marker compounds. 13C-octanoic acid is suitable for understanding the kinetics of solids, and 13C-acetic acid is suitable for understanding the kinetics of liquids. Test meals made of egg yolk mixed with 13C-octanoic acid and baked are mainly used as solid test meals for mice. The hurdles to performing the breath test are lower than those for scintigraphy because these materials are not radioactive and easy to handle. The breath test may be the most suitable method for analysis over time, in which repeated measurements are performed in the same animal. There is a great need for longitudinal studies that repeatedly measure gastric emptying, and breath tests are useful for tracking changes in gastric emptying over time as a disease, such as diabetes, progresses [38,39,40]. The breath test in mice is a useful method that contributes to our understanding of the pathophysiology of gastric emptying at each disease stage and the therapeutic effects of drugs.

The active use of these noninvasive methods is an effective approach to streamline the compatibility of data obtained from basic research with clinical data. This approach is useful for the development of new noninvasive methods. Other methods of noninvasively assessing gastric emptying in mice include fluoroscopy [41], near-infrared fluorescence reflectance imaging [42, 43], bioluminescence imaging [44], ultrasonography [45] and multispectral optoacoustic tomography [46]. These imaging techniques allow visualization of changes in gastric emptying. The advantage of this was not observed in the breath test. Polysaccharides, such as pectin, can affect intestinal migration and absorption, in which case gastric emptying cannot be accurately measured with breath tests. Imaging techniques can be useful for detecting changes in gastric emptying caused by the physical properties of the ingested material. Advances in methodologies with noninvasive approaches will bridge the gap in data compatibility between humans and mice.

Small Intestinal Motility

Noninvasive methods for assessing small intestinal motility in humans include the hydrogen breath test [47], WMC test [48], scintigraphy [49], and magnetic resonance imaging (MRI) [50,51,52]. In contrast to gastric emptying, these techniques are often used in research, but have yet to be fully applied in clinical practice. The normal range in the assessment of small intestinal motility is wide, and further studies are needed to standardize the data objectively.

Similarly, effective noninvasive methods are limited to the evaluation of small intestinal motility in mice. Therefore, invasive methods have been frequently used in recent years, and small intestinal motility is measured as small intestinal transit, which is the distance traveled in the gastrointestinal tract by orally administered non-absorbable dyes [53,54,55,56]. Imaging techniques such as fluoroscopy [57, 58] and fluorescence imaging [59, 60] are used as noninvasive methods to evaluate small intestinal motility in mice. These methods determine small intestinal motility by visualizing peristalsis and segmental movements. Recently, a method for quantitative analysis of small intestinal motility using ultrasonography has been developed in mice (Fig. 1), which enables noninvasive and rapid quantification of small intestinal motility [61]. Additionally, the usefulness of ultrasonography has been validated in animals other than mice [62,63,64]. Ultrasonography is an in vivo imaging modality that can be easily performed in both humans and animals and is expected to contribute to translational research.

Fig. 1.

Noninvasive evaluation of changes in intestinal motility using real-time ultrasonography in mice. (A) Abdominal ultrasonography in mice was performed using a digital micro-ultrasound system (Vevo 3100, FujiFilm VisualSonics, Toronto, Canada) with a 55-MHz linear array transducer. (B) Representative images of the intestine with trace lines (green) drawn for the quantification of intestinal motility. For the analysis of changes in the short axis, trace lines were placed on the boundary between the intestinal wall and lumen. (C) Representative images of the change in speckle points on trace lines. Locomotion activity of the intestinal wall was defined as the average displacement and strain rate between any number of speckle points on the trace line for all time frames. (D) Quantification of locomotion activity. These parameters were calculated by converting the maximum rate of change (ΔL) calculated from the difference between the minimum length (L0) and maximum length (L) between the two points to a unit of time. (E) Representative examples of small intestinal motility (long-axis) as assessed using ultrasonography. White triangles indicate the expansion site of the lumen. Dysmotility was induced by the antidiarrheal drug, loperamide. Mice were subcutaneously administered loperamide dissolved in saline at a dose of 5 mg/kg. Quantification of locomotion activity was calculated from the change in speckle points on the trace lines (n=6, **P<0.01; significantly different from the control). Reproduced with permission from Kishi et al. [61, 74]. The animal protocols of these studies were approved by the Institutional Review Board of The University of Tokyo (No. P18-131).

However, these noninvasive methods have limitations. The measurements need to be performed under anesthesia in small animals, such as mice. We must understand that the conditions of the tests are different in humans and mice because general anesthesia may affect GI motility. Despite these limitations, these imaging techniques are useful as noninvasive methods to investigate small intestinal motility in mice and can be combined with tests of small bowel transit to provide accurate assessment.

Colonic Motility

In vivo evaluation is essential to investigate changes in colonic motility affected by diseases or drugs; however, there are few appropriate methods for the assessment of murine colonic motility. Hence, knowledge of the physiology of colonic motility in vivo is lacking.

A bead expulsion assay is commonly used to quantitatively assess murine colonic motility as a noninvasive technique. In this assay, glass beads are inserted approximately 2–3 cm into the rectum under anesthesia, and the time taken for the beads to exit the anus reflects the colonic transit time. This method has been used in many studies and is now a model for in vivo studies to understand the physiology of colonic motility [13,14,15, 55, 65, 66]. On the other hand, scintigraphy and radiopaque markers have been used to measure colonic transit time in humans as a method that can provide accurate and quantitative results [67, 68]; there is a gap in measurement technology between humans and mice. Additional studies are needed to determine whether these tests and colonic motor patterns in humans correlate with those in mice. Therefore, although data compatibility in humans and mice must be carefully determined, the bead expulsion assay is a useful noninvasive tool that can easily assess colonic transit in mice.

In addition to the bead expulsion assay, observation of the number and shape of feces has been applied as a simple noninvasive method to indirectly assess murine colonic motility [69]. This method provides markers for predicting abnormalities in colonic function, such as diarrhea and constipation; however, previous reports showed that clinical markers such as stool morphology and frequency of defecation do not adequately reflect colonic transit time in humans [70]. This discrepancy is also true in mice, and a report showed that colonic transit time was significantly delayed in colitic mice with diarrhea [71]. These findings suggest that information about colonic motility obtained from clinical markers is limited, and that accurate assessment of colonic motility requires physiological measurements.

Noninvasive techniques using colonic manometry and ultrasonography have been developed as methods to physiologically evaluate colonic motility in mice. Colonic manometry is a method for evaluating the contraction pattern of colonic motility using a manometer inserted into the colon [72, 73]. This method is useful for assessing drug- and disease-induced changes in murine colonic motility in a conscious state. Ultrasonography is a diagnostic imaging technique used to assess the status of an organ. The advantage of ultrasonography is that dynamic and morphological assessments of GI motility can be performed simultaneously in real time. Recently, a report proposed an analytical method for the quantitative detection of colonic motility disorders using ultrasonography in mice (Fig. 1) [74]. Ultrasonography can objectively obtain supplementary information on the function of peristalsis in the colon. The data obtained from these methods may improve our ability to assess colonic motility in mice. On the other hand, they focus on the evaluation of contraction and peristalsis in the colon and are indirect methods for revealing the propulsive force of the contents in the colon.

Taken together, there is an ongoing need for the development of novel noninvasive methods to assess the function of colonic motility in mice. Variations in measurement techniques will contribute to the development of new drugs and a comprehensive understanding of GI diseases.

Future Perspectives

The data obtained from experiments with mice should contribute to improved reproducibility in other animals, including humans. Considering the similarity of cellular mechanisms, it is expected that there are similarities in GI motility patterns between humans and mice. However, at present, experimental conditions and measurement methods are very different between humans and mice; the development of a noninvasive method applicable to both humans and mice is required to further improve data compatibility.

In vivo imaging techniques used in clinical practice, such as MRI, may also be useful in preclinical studies in mice. In recent years, there has been rapid development in MRI-based GI motility assays in humans [75, 76] and rats [77, 78]. MRI-based imaging techniques are a new development worthy of attention as they allow visual monitoring of GI wall motion through time-resolved images. This method has no risk of radiation exposure and does not limit frequent measurements for longitudinal observations. Evidence for the utility of MRI is building in both basic and clinical areas, as supported by a number of studies. Future studies with a view of applying it to mice are attractive and valuable in translational or reverse translational research. Advances in noninvasive imaging techniques for assessing GI motility will reveal commonalities in GI function in humans and mice, which helps to strengthen the correspondence between the respective data.


Noninvasive methods for measuring murine GI motility are essential tools that contribute to our understanding of GI physiology in vivo and to guidelines for the ethics of animal research. In particular, imaging techniques such as diagnostic imaging are advancing rapidly in clinical practice, and the idea of reverse-translating these into noninvasive techniques for mice will be a great asset for preclinical research. Applying clinically used techniques to obtain basic data from mice allows for smooth extrapolation to clinical practice, which may improve reproducibility in human and veterinary practice. We hope that knowledge of these noninvasive techniques has spread throughout the field of laboratory animal science, benefiting both animals and researchers.

Conflicts of Interest

The authors declare no conflicts of interest.


This work was supported by a Grant-in-Aid for JSPS Fellows (20J11240 to K.K.) and Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (24248050 to M.H.).

Author Contributions

K.K. drafted the manuscript; K.K., N.K., and M.H. edited and revised the manuscript; K.K., N.K., and M.H. approved the final version of the manuscript.


Yoshiharu Tsuru (Primetech Life Science Laboratory, PRIMETECH Corporation) provided images related to ultrasonography in this review.

© 2021 Catalyst Unit

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