As an instrument for measuring body composition in experimental animals, dual energy X-ray absorptiometry (DXA) is ideal for accuracy, cost, and measurement efficiency. However, there is too little insight into the effectiveness of the various aspects of applying DXA to experimental animals. We investigated whether to compare and verify the precision and accuracy of DXA and nuclear magnetic resonance (NMR) animal body composition analyzers.
We used 30 Institution of Cancer Research mice in the study. First, in order to evaluate the reproducibility of DXA and NMR, we did repeated measurements by repositioning each mouse in anesthesia and euthanasia states. Subsequently, the accuracy of each device was evaluated by comparing the weight measured before the experiment, the weight of the tissue extracted from the mice after the experiment, and the measured DXA and NMR. In addition, when measuring the body composition of animals, we compared the time and the measurable body composition parameters and summarized the advantages and disadvantages of the 2 devices.
Compared to NMR, DXA had the advantage of a fast measurement of bone composition and rapid image analysis. In addition, DXA showed a higher correlation (>95%) with fat mass, lean mass baseline than did NMR (>85%).
In conclusion, DXA was confirmed to have higher precision and measurement accuracy than did NMR. Therefore, DXA is an effective method for evaluating the body composition of experimental animals.
Animal clinical trials are conducted in various ways in order to verify the effects of various physical activities of exercise on health as well as for research on the treatment of diseases and the efficacy of food. In addition, animal clinical trials are a procedure that verifies the safety of new drugs or treatments before humans are exposed to new molecular entities.[
Methods of measuring body composition in human clinical practice include underwater weighing [
In animal studies, the underwater weighing used in human clinical studies has limitations in measuring bone density and region of interest (ROI), because it can only distinguish between body FM and lean body mass (LM). In addition, in the case of air displacement plethysmography, it is difficult to realize the sensitivity to measure the air displacement of an animal because of the sensitivity to measure the air displacement. Skinfold thickness method cannot be applied to animals because it relies on the calculation formula from human body data accumulated through various studies. Since electrical bioimpedance analyses focus on people’s convenience rather than accuracy, they have little value to apply to animals. Computed tomography (CT) can also be considered as a method of measuring animal body composition. However, the radiation exposure of the experimenter is more than 150-fold lager than that of DXA, and there is no advantage compared to DXA or NMR in terms of cost to use it to measure animal body composition.[
Like human body composition measuring, animal body composition measuring must be able to identify factors such as body weight, body fat, BMC, and the cost and accuracy of the measurement must also be considered. Considering these factors, DXA and NMR may be candidates for the most suitable equipment for measuring body composition in animals. Nevertheless, studies to select the “gold standard” equipment for measuring body composition in animals by DXA and NMR are insufficient. In particular, the only study comparing the accuracy of DXA and NMR in mice focused on comparing of body FM and LM, but bone mineral density (BMD) and BMC were not analyzed.[
In this study, the accuracy of DXA was measured in measuring BMD and BMC (NMR could not be compared because bone and mineral do not contribute to the NMR signal),[
A total of 30 mice were used in the experiment (10 mice/group), and a body weight target was set for each mouse group to start the experiment. In the breeding stage before the experiment, 12 candidate mice were reared for each group, and the body weight was monitored through an electronic scale. When the target body weight was reached, it was used in the experiment, and 2 animals were excluded per group. The main purpose of this study is to compare the accuracy of body composition values measured by DXA and NMR. Therefore, the FM and LM measurements of DXA and NMR were compared with the weight of the dissected tissue (autopsy). In addition, the reproducibility of each equipment was confirmed from the coefficient of variation (CV) of the measurement item.
We purchased male Institution of Cancer Research mice (n=30) from Orient Bio (Seongnam, Korea). We provided all animals with
When measuring DXA (iNSiGHT VET DXA; OsteoSys, Seoul, Korea) or NMR (EchoMRITM-700; EchoMRI, Houston, TX, USA), there are a total of 2 measuring conditions for animals, which are defined as euthanasia with repositioning (RE), anesthesia with repositioning (RA). In the initial preparation of all animals, the weight is measured once using an electronics scale. In addition, this value is defined as a reference value of weight for accuracy verification by measuring it once more on the day of the experiment. When measuring DXA, the mouse’s abdomen was positioned downward in the center of the measuring plate, and the limbs were placed in a lightly straight outward direction. During repositioning, the mouse was completely separated from the measuring plate, and then the mouse was positioned in the same way as before according to the scale printed on the measuring plate. In NMR measurement, the mouse is pushed all the way into the cylinder and a fixing device is inserted, but there is a margin to prevent the mouse from being compressed. When repositioning, after removing the mouse from the cylinder, positioning was performed in the same way before.
On the day of the experiment, we measured 7 times per all mice under both anesthesia and euthanasia conditions. We repositioned animals at the initial 3 measurements, and posture at the 3rd measurement was maintained for the next 4 measurements. After relocating the animals in the initial 3 measurements, the posture was maintained during subsequent measurements. The time required for one measurement of DXA equipment was measured with a stopwatch. The time required was defined as the time until the operation was completed after pressing the measurement start button of the equipment software. We used the DXA equipment to collect FM, LM) body weight, BMD, BMC, and fat percentage of the entire body range. In addition, we collected left femur BMC (lfBMC) and femur BMC (fBMC) by designating the femur region as a local ROI value. Finally, we collected images provided by DXA. All DXA image shooting was done using “mouse mode” in Insight software (Version 1.0.6; OsteoSys, Seoul, Korea). The X-ray tube settings in “mouse mode” were 60 kV/0.8 mA and 80 kV/0.8 mA at low energy and high energy, respectively.
On the day of the experiment, we measured all mice 3 times each by NMR after anesthesia. We repositioned the animals at every measurement. On the day of the experiment, we measured all mice 3 times each by NMR after euthanasia. We repositioned the animals at every measurement. We measured the time required for one measurement with the NMR equipment with a stopwatch. The time required was defined as the time until the operation was completed after pressing the measurement start button of the equipment software. We collected FM, LM, and body water over the entire body range using NMR.
After we completed the measurement with all equipment, the mouse was autopsied, and after we extracted the main tissue, the mouse was weighed by using an electronic scales (Sartorius Entris® 124-1S; Sartorius Corp., Göttingen, Germany). We combined the weights of kidney adipose tissue, epididymal adipose tissue, intestine adipose tissue, and subcutaneous fat, and defined it as a reference value of FM for accuracy verification. The sum of the measurements of left femur and right femur was defined as femur mass and as the reference value of fBMC for accuracy verification. We selected one mouse from each group (low-weight, moderate-weight, and high-weight groups) and measured the time required for autopsy, which was defined as the time from the moment of taking the action necessary to autopsy the euthanasia animal to the completion of weighing for all items.
To evaluate the precision, we calculated the CV of each item in the repeated measurement data. The mean±standard deviation (SD) of the CV values for each measurement is summarized according to the mouse condition. In order to evaluate the accuracy, we summarized average residual values for each measurement
We derived the CV of body composition measurement results according to the measuring conditions of all mice (
In order to confirm the error of measurement according to the body weight, the weight was divided in the 3 grades; the measured CV is shown in
We compared the measurements of each body component according to the measuring conditions with the reference values (
DXA is a method of deriving body composition results using the difference between high-energy X-ray images and low-energy X-ray images.[
Compared to X-ray or CT, NMR has no radiation exposure. Its image contrast and resolution has excellent advantages for soft-tissue and brain examination,[
In order to reduce the errors in measuring the experimental animals, what is most important is the degree of fixation at the time of measurement. It is natural that accuracy is higher in euthanasia than in anesthesia, because under anesthesia, there is a high possibility of errors caused by differences in fine movements that depend on the degree of breathing and level of anesthesia. In general, it is known that DXA is less capable of scanning non-anesthetized animals compared to NMR.[
In previous study, it was mentioned that the measurement speed of NMR has an advantage over DXA.[
The precision of FM and LM in DXA was higher than that of NMR (
FM had an error of less than about 1 g compared to the reference value and showed a correlation of more than 95%. The error of NMR was +2.08 g, which was larger than that of DXA. Since NMR showed a correlation of up to about 92% with the reference value, we concluded that DXA has higher estimation accuracy for the reference value than does NMR. When compared to the actual mouse body weight, DXA measured about 2.04% to 2.96% lower. However, we found that the body weight difference between individuals was measured with an accuracy of 99% or more (
The
To sum up, DXA exhibits high accuracy with little difference from the actual autopsy weight of FM. Because of this FM’s accuracy, LM can also be accurately measured. However, BMD and BMC were different from the autopsied weight. This difference in weight is due to the difference in water content of bone, and BMD, BMC between individuals can be compared relatively accurately with X-ray images.
NMR has a longer measurement time than DXA, but has the advantage of being able to measure without anesthesia even when the animal is alive. In addition, NMR equipment can measure water in addition to fat and lean. However, since there are body tissues that cannot be measured, because they do not respond well in the NMR method, such as bone, NMR contains a fundamental error in weight estimation. In addition, there is a disadvantage in that visual information cannot be acquired, because an image of the measured animal is not provided separately.[
Unlike NMR, DXA can measure BMD and BMC, and it is judged to be suitable as a “gold standard” for grasping the body composition of animals because of its high precision and accuracy of measurement. In particular, if the experimental animals are deeply anesthetized to minimize movement during DXA measurement, the effect on the measurement results seem to be insignificant.
This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ014155052019)” Rural Development Administration, Republic of Korea.
The entire experiment was done in accordance with the guidelines of the Animal Experimental Ethics Committee of the Agency for Korea National Food Cluster (approval number: KNFC-IACUC-20-001).
No potential conflict of interest relevant to this article was reported.
Scatter plot of correlations between measurement results and reference values of dual energy X-ray absorptiometry (DXA) or nuclear magnetic resonance (NMR). RE, euthanasia with repositioning; RA, anesthesia with repositioning; FM, fat mass; fBMC, femur bone mineral content.
Measurement images provided by dual energy X-ray absorptiometry used in this study.
Results of dual energy X-ray absorptiometry and nuclear magnetic resonance according to repeated measures
DXA (n=30) | NMR (n=30) | |
---|---|---|
FM (CV) | ||
RE | 3.88±2.53 | 17.00±12.91 |
RA | 5.05±3.58 | 14.96±10.46 |
| ||
LM (CV) | ||
RE | 0.38±0.18 | 3.91±2.39 |
RA | 0.43±0.25 | 2.75±1.70 |
| ||
Body weight (CV) | ||
RE | 0.27±0.15 | NA |
RA | 0.41±0.34 | NA |
| ||
Body water (CV) | ||
RE | NA | 26.66±43.77 |
RA | NA | 13.65±29.73 |
| ||
BMC (CV) | ||
RE | 2.36±1.44 | NA |
RA | 2.90±1.85 | NA |
| ||
lfBMC (CV) | ||
RE | 6.38±3.73 | NA |
RA | 7.70±5.95 | NA |
The data is presented as the mean±standard deviation.
DXA, dual energy X-ray absorptiometry; NMR, nuclear magnetic resonance; FM, fat mass; CV, coefficient of variation; RE, euthanasia with repositioning; RA, anesthesia with repositioning; LM, lean mass; BMC, bone mineral content; lfBMC, left femur bone mineral content; NA, not applicable.
Results of dual energy X-ray absorptiometry and nuclear magnetic resonance according to repeated measurements classified by body weight
RE | RA | |||
---|---|---|---|---|
|
| |||
DXA | NMR | DXA | NMR | |
Low-weight (n=10) | ||||
FM (CV) | 5.63±2.90 | 27.03±14.69 | 6.92±4.72 | 19.69±13.08 |
LM (CV) | 0.36±0.21 | 6.11±2.17 | 0.44±0.32 | 2.75±1.93 |
BMC (CV) | 3.51±1.56 | NA | 3.51±1.56 | NA |
| ||||
Mid-weight (n=10) | ||||
FM (CV) | 3.47±2.01 | 6.11±2.17 | 5.48±2.58 | 15.28±8.69 |
LM (CV) | 0.41±0.17 | 2.51±1.49 | 0.42±0.25 | 2.75 ±1.93 |
BMC (CV) | 1.70±1.08 | NA | 2.47±2.11 | NA |
| ||||
High-weight (n=10) | ||||
FM (CV) | 2.54±1.61 | 8.29±4.29 | 3.17±2.58 | 8.29±4.29 |
LM (CV) | 0.38±0.15 | 2.91±1.49 | 0.44±0.21 | 2.66±1.70 |
BMC (CV) | 1.86±0.89 | NA | 2.47±2.11 | NA |
The data is presented as the mean±standard deviation.
RE, euthanasia with repositioning; RA, anesthesia with repositioning; DXA, dual energy X-ray absorptiometry; NMR, nuclear magnetic resonance; FM, fat mass; CV, coefficient of variation; LM, lean mass; BMC, bone mineral content; NA, not applicable.
Correlation between measurement results and reference values of dual energy X-ray absorptiometry and nuclear magnetic resonance
DXA (n=30) | NMR (n=30) | Reference | |
---|---|---|---|
FM | 1.63±1.51 | ||
RE | 0.61 (0.959) | 2.08 (0.851) |
|
RA | 0.49 (0.965) | 1.63 (0.916) |
|
| |||
Body weight | 29.36±10.16 | ||
RE | −0.80 (0.996) | NA | |
RA | −0.60 (0.996) | NA | |
| |||
lfBMC | 0.133±0.060 | ||
RE | −0.108 (0.954) | NA | |
RA | −0.108 (0.961) | NA |
The data is presented as mean signed difference (
Electronic scale.
DXA, dual energy X-ray absorptiometry; NMR, nuclear magnetic resonance; FM, fat mass; RE, euthanasia with repositioning; RA, anesthesia with repositioning; lfBMC, left femur bone mineral content; NA, not applicable.