This article describes the deuterium oxide dilution technique in two mammals, an insectivore and carnivore, to determine total body water, lean body mass, body fat mass, and water consumption.
Body condition scoring systems and body condition indices are common techniques used for assessing the health status or fitness of a species. Body condition scoring systems are evaluator dependent and have the potential to be highly subjective. Body condition indices can be confounded by foraging, the effects of body weight, as well as statistical and inferential problems. An alternative to body condition scoring systems and body condition indices is using a stable isotope such as deuterium oxide to determine body composition. The deuterium oxide dilution method is a repeatable, quantitative technique used to estimate body composition in humans, wildlife, and domestic species. Additionally, the deuterium oxide dilution technique can be used to determine the water consumption of an individual animal. Here, we describe the adaption of the deuterium oxide dilution technique for assessing body composition in big brown bats (Eptesicus fuscus) and for assessing water consumption in cats (Felis catis).
Body condition scoring systems and body condition indices are common techniques used for assessing the health status or fitness of a species1,2. Many domestic and zoological species have unique body condition scoring (BCS) systems that are used to assess an animal's muscle and superficial fatty tissue3. However, BCS assessment relies upon the evaluator—meaning that BCS is an objective or semiquantitative measurement when assessed by a trained evaluator. In wildlife species, body condition indices are commonly used rather than BCS and are based upon a ratio of body mass to body size or body mass to forearm2. Body condition indicis are often confounded by the effects of foraging and can be confounded by body size as well as statistical and inferential problems4.
An alternative to body condition scoring systems and body condition indices is using a stable isotope to determine body composition. One commonly used stable isotope is deuterium oxide (D2O), a non-radioactive form of water in which the hydrogen atoms are deuterium isotopes. The deuterium oxide dilution method described in this study can be a non-subjective, quantitative, and repeatable technique used to estimate body composition in humans5 and a wide range of species4,6,7. This technique can be advantageous for studying the body composition in wildlife. For example, it can be used to assess longitudinal changes in body composition, such as before and after a management action. However, in some wildlife species deuterium oxide can overestimate the actual water content8. Therefore, when adapting the technique for a species, it is important to validate the method by comparing the deuterium oxide method to carcass analysis for non-endangered species. For threatened and endangered species, a non-destructive method such as dual x-ray absorptiometry (DXA) should be considered as an alternative comparison method to the gold-standard destructive method of complete carcass analysis.
In addition to body composition, the D2O dilution technique can be used to determine the water consumption of an individual animal9. This unique application of D2O can be used to answer not only research questions, but can be useful for assessing the water consumption of individual animal(s) housed in large social settings.
Here, we describe the adaption of the D2O dilution technique for assessing body composition in an insectivore, big brown bats (Eptesicus fuscus), and for assessing water consumption in a carnivore, cats (Felis catis).
All experiments described here were approved by the University of Missouri Animal Care and Use Committee and conducted under the Missouri Department of Conservation (MDC) Wildlife Scientific Collection permit (Permit #16409 and #17649).
1. Preparation of sterile, isotonic, salinated D2O stock solution
2. Preparation of sterile, isotonic, salinated D2O stock working solution for bats
3. Determination of body composition of big brown bats (Eptesicus fucsus) with D2O
NOTE: The stock solution of D2O used in the protocol is 0.1598 g/mL. Before collecting blood, ensure that removing up to 200 µL of blood will be ≤ 10% of the total blood volume of the bat and is within the Institutional Animal Care and Use Committee's (IACUC) established guidelines for blood collection. All animals should be fasted or abdomen palpated to ensure an empty stomach. A recent meal could alter the animal's weight resulting in confounded results since calculations for determining body fat rely upon the body mass of the animal.
4. Fourier-transform infrared spectrophotometry analysis
5. Calculation of body composition
6. Determination of water composition in a carnivore (Felis catus, domestic cat)
The deuterium oxide dilution technique can be used to assess the body composition of a variety of species. To demonstrate the adaptability, we are reporting the first use of the deuterium oxide dilution technique in a North American insectivorous bat species, Eptesicus fuscus, the big brown bat for representative results. A timing plateau was completed by taking pre- and post-D2O injection blood samples as should be done with any species where the equilibration period is unknown. It was determined that two hours post-injection in non-torpid bats was adequate for equilibration. With the equilibration time known, the total body water, lean body mass, and body fat mass for 13 wild-caught big brown bats and 8 captive big brown bats were determined (Table 2). An additional 2 wild-caught big brown bats and 5 captive big brown bats were determined to have a negative body fat mass. A negative body fat mass is calculated due to one or more of the following reasons: not receiving the entire dose of deuterium oxide, becoming torpid during the equilibration phase, having abnormally large fat masses and minimal lean mass, or bats having under 3%−5% body fat as determine by DXA (Table 3).
White-nose syndrome has caused many bat species to decline, so the technique was compared to the body fat measured using DXA. Figure 1 shows the percentage of body fat determined by the D2O dilution technique and DXA (n = 19). The two techniques were well correlated with a Pearson's r = 0.897 (Figure 2) and were not statistically different (one-way analysis of variance (ANOVA), F-value = 0.366, p = 0.549). The body fat showed strong correlations between body fat and body weight (Figure 3). The D2O dilution technique did not consistently over or underestimate the body fat mass.
The deuterium oxide method has been previously validated in cats16. Table 4 shows an example of the total body water, lean body mass, and body fat mass of a single cat9. Hooper et al.9 was the first to report the use of deuterium oxide dilution to measure the water consumption of socially housed animals with the daily water consumption of the cats during each dietary block of the experiment, as shown in Figure 4.
Figure 1: Deuterium oxide and DXA line plot. Each point represents the body fat percentage of an individual bat as determined by DXA or deuterium oxide. The mean is the light green point with error bars indicating the standard error of the mean. Please click here to view a larger version of this figure.
Figure 2: Percentage of body fat in big brown bats. Deming regression (solid blue line, Pearson's r = 0.897) comparing the percentage of body fat determined by DXA (x-axis, the reference method) and the percentage of body fat determined by deuterium oxide (y-axis, the test method) in big brown bats with 95% confidence intervals designated by gray shading. The green dash identity line drawn represents the regression line when the methods are equal. Please click here to view a larger version of this figure.
Figure 3: Percentage of body fat in big brown bats compared to body weight. Body weight of each bat plotted against the body fat percentage determined by D2O or DXA. A strong correlation exists between the body weight and body fat as determined by DXA (dark blue line, Pearson's r = 0.88) and D2O (blue line, Pearson's r = 0.86). Please click here to view a larger version of this figure.
Figure 4: Water consumption of socially housed cats. Representative results of the daily water consumption of socially housed cats during an experiment evaluating the effects of dietary constituents on water consumption. This figure has been modified from Hooper et al.9. Please click here to view a larger version of this figure.
Parameter | Setting |
Number of scans | 64 |
Resolution | 2 |
Data spacing | 0.946 cm-1 |
Final format | Absorbance |
Correction | None |
Use fixed Y-axis limits in collection window | Min -0.01, Max 0.03 |
Bench range | Max 6.38, Min -5.02, Loc 1024 |
Total absorbing peak sensitivity | 50 |
fringes or channeling sensitivity | 80 |
Derivative peaks sensativity | 51 |
Baseline error sensitivity | 50 |
CO2 levels sensitivity | 19 |
H2O levels sensitivity | 19 |
Apodization mode | Happ-Genzel |
Phase correction | Mertz |
Filters set based upon | velocity |
low pass filter | 11,000 |
high pass filter | 20 |
Table 1: Spectral software settings. Parameter settings used for spectral recording software.
Animal | Species | Body weight (kg) |
D2O injected (g) |
Total body water (g) |
Lean body mass (g) |
Body fat mass (g) |
Body fat mass (%) |
DXA lean + bmc (g) |
DXA fat (g) |
DXA fat (%) |
1 | Eptesicus fuscus | 0.01715 | 0.0740 | 11.80 | 16.15 | 1.00 | 5.80 | 14.65 | 0.75 | 4.80 |
2 | Eptesicus fuscus | 0.01950 | 0.0920 | 13.80 | 18.83 | 0.69 | 3.50 | 16.20 | 1.40 | 7.90 |
3 | Eptesicus fuscus | 0.01677 | 0.08 | 11.33 | 15.47 | 1.30 | 7.74 | 11.33 | 1.30 | 7.74 |
4 | Eptesicus fuscus | 0.02129 | 0.097 | 12.51 | 17.09 | 4.20 | 19.7 | 15.9 | 19.65 | 19.2 |
Table 2: Body composition of big brown bats. The representative results of total body water, lean body mass, and body fat as determined by deuterium oxide dilution in big brown bats are shown in columns 5−8. Representative results of the lean body mass plus bone mineral content and body fat as determined by DXA in the same big brown bats are shown in columns 9−11.
Animal | Species | Body weight (kg) |
D2O injected (g) |
Total body water (g) |
Lean body mass (g) |
Body fat mass (g) |
Body fat mass (%) |
DXA lean + bmc (g) |
DXA fat (g) |
DXA fat (%) |
Comment |
1 | Eptesicus fuscus | 0.0277 | 0.1299 | 34.18 | 46.69 | -19.02 | -68.74 | 9.90 | 26.55 | 62.80 | Equili-bration time insufficient |
2 | Eptesicus fuscus | 0.0185 | 0.0810388 | 64.23 | 87.75 | -69.25 | -374.33 | 14.20 | 17.30 | 17.95 | Full dose not injected |
3 | Eptesicus fuscus | 0.0164 | 0.0719 | 17.38 | 23.74 | -7.33 | -44.68 | 14.15 | 14.40 | 1.70 | Less than 3% fat |
4 | Eptesicus fuscus | 0.0212 | 0.0994 | 54.57 | 74.54 | -53.37 | -252.0 | 16.41 | 19.01 | 13.65 | Bat became torpid (cool to touch) |
Table 3: Body composition of big brown bats. Representative results from bats that did not receive the entire dose of deuterium oxide, became torpid during the equilibration phase, bats with abnormally large fat mass and minimal lean mass, or bats under 3%−5% body fat as determine by DXA. The representative results of total body water, lean body mass, and body fat as determined by deuterium oxide dilution are shown in columns 5−8. Representative results of the lean body mass plus bone mineral content and body fat as determined by DXA are shown in columns 9−11.
Block | Species | Body weight (kg) |
D2O injected (g) |
Total body water (kg) |
Lean body mass (kg) |
Body fat mass (kg) |
Body fat mass (%) |
Daily water consumption (mL/day) |
Dietary Treatment |
1 | Felis Catus | 4.830 | 3.36 | 2.69 | 3.68 | 1.149 | 23.8 | 96.8 | Control |
2 | Felis Catus | 4.764 | 3.45 | 2.66 | 3.63 | 1.136 | 23.8 | 217.5 | High Moisture |
3 | Felis Catus | 4.727 | 3.25 | 2.50 | 3.41 | 1.314 | 27.8 | 125.1 | High Selenium |
Table 4: Body composition and water consumption in a single feline. Representative results of deuterium oxide dilutional technique for assessing the lean body mass, fat mass, and water consumption of one cat at three different time points during the study conducted by Hooper et al.9.
The use of deuterium oxide to determine TBW has been used since the 1940s17 and is used in humans and a variety of domestic and wildlife species4,6,7. Other non-destructive techniques have been developed including bioelectrical impedance analysis (BIA), DXA, and quantitative magnetic resonance (QMR). Each method has advantages and disadvantages that should be considered before selecting a particular methodology for assessing body composition. This protocol selected to use DXA as a comparison method for deuterium oxide to assess body composition, because the equipment is available as a core university resource with minimal cost, minimal time is required per scan (30 s per bat), and it is not sensitive to variables such as body temperature and skin insulation.
When adapting the deuterium oxide dilution technique to a species of interest, a pilot study should be initiated to determine the time required for equilibration18. This can be done by taking a background sample, and a blood sample every 15 minutes post-injection. For small species such as bats, several bats can be bled at the different time intervals instead of a single animal18. The equilibration time can change when animals, such as bats, go into torpor, which explains why some of our animals had a negative percent body fat (Table 3). If a negative percent body fat is obtained, and the deuterium dose had sufficient time to fully equilibrate with the animal's body water, then it is likely the dose was not completely injected. Because the deuterium oxide dilution technique is highly dependent upon the full dose being administered and accurate recording of the amount of deuterium injected, this technique should only be completed by individuals skilled in performing injections. Additionally, anesthetizing or sedating animals can assist with ensuring the entire dose can be administered.
When administering the deuterium oxide, it is important to determine an appropriate concentration to administer to the animal. Using a 0.7 g/kg dose for the cats, the stock solution concentration was appropriate, whereas for the big brown bats a 0.75 g/kg dose required the stock solution of deuterium oxide to be diluted. When diluting the stock solution, an isotonic solution such as 0.9% NaCl should be used. To avoid altering the total body water of small mammals, dilute the dose of deuterium oxide as minimally as possible, just enough to ensure the dose can be measured accurately.
The doses presented here are detectible using FTIR spectrometry. FTIR spectrometry is less expensive and easier to maintain, but not as sensitive as isotope ratio mass spectrometry (IRMS)19,20. FTIR spectrometry can be used to measure deuterium enrichment in plasma and saliva, but it is not recommended to use an FTIR transmission cell to analyze deuterium enrichment in urine19. If urine is the desired sample type, then an attenuated total reflection (ATR) attachment should be used with the FTIR or IRMS should be used to assess deuterium enrichment for calculation of TBW19.
Additionally, the doses used for the cats were adequate to allow detection of deuterium oxide 14 days post-injection. Because the concentration of the deuterium oxide 14 days post-injection was detectable, the water consumption of the cats could be calculated (Figure 4). This innovative use of deuterium oxide can be employed in field studies to measure body water turnover for species with high recapture rates or for animals housed in groups in ex situ or laboratory studies. However, before employing in field studies, researchers must assess if the animal can be captured and held for the duration of the equilibration period. This prolong handling period is one of the disadvantages of the deuterium oxide technique and could be problematic as many endangered species permits limit the duration that a particular animal can be held. Additionally, animals cannot have recently eaten as the washout technique relies upon the measurement of body mass; therefore, a recent meal can confound the results. An additional consideration is whether an animal must be anesthetized or sedated for subcutaneous injection and blood collections or if the animal can be restrained without sedation/anesthesia. It has been suggested that the rate of body water turnover could be a significant indicator for human health21. The increased water consumption in cat 5 (Figure 4) was documented before traditional biochemical marks of renal failure, and concentrations of creatinine and blood urea nitrogen (BUN) were elevated, suggesting that body water turnover could also be an indicator of health in animals.
The authors have nothing to disclose.
This research was supported by MDC Cooperative Agreement (#416), US Forest Service Cooperative Agreement (16-JV-11242311-118), American Academy of Veterinary Nutrition and Waltham/Royal Canin, USA Grant (grant number: 00049049), NIH training grant (grant number: T32OS011126), and the University of Missouri Veterinary Research Scholars Program. The authors thank Shannon Ehlers for pre-reviewing this manuscript. We thank Dr. Robert Backus for providing the D2O standards and allowing use of his laboratory.
0.2 micron non-pyrogenic disk filter | Argos Technologies | FN32S | nylon, 30mm diameter, 0.22um, sterile |
1.5 mL conical microcentrifuge tubes | USA Scientific | 1415-9701 | 1.5 ml self-standing microcentrifuge tube, natural with blue cap |
10 mL sterile glass vial for injection | Mountainside Medical Equipment | MS-SEV10 | clear, sterile glass injection unit |
10 mL syringe | Becton Dickinson | 305219 | sterile 10 mL syringe individually wrapped |
100 mL sterile glass vial for injection | Mountainside Medical Equipment | AL-SV10020 | clear, sterile glass injection unit |
20 gauge needle | Exel | 26417 | needles hypodermic 20g x 1" plastic hub (yellow) / regular bevel |
22 gauge needle | Exel | 26411 | needles hypodermic 22g x 1" plastic hub (black) / regular bevel |
deuterium oxide | Sigma-Aldrich | 151882-25G | 99.9 atom % D |
isofluorane | Vetone | 3060 | fluriso isoflurane, USP |
OMNIC Spectra Software | ThermoFisher Scientific | 833-036200 | FT-IR standard software |
petroleum jelly | Vaseline | 305212311006 | Vaseline, 100% pure petroleum jelly, original, skin protectant |
plastic capillary tubes | Innovative Med Tech | 100050 | sodium heparin anticoagulant, 50 μL capacity, 30 mm length |
Sealed liquid spectrophotometer SL-3 FTIR CAF2 Cell | International Crystal Laboratory | 0005D-875 | 0.05 mm Pathlength |
sodium chloride | EMD Millipore | 1.37017 | suitable for biopharmaceutical production |
Thermo Electron Nicolet 380 FT-IR Spectrometer | ThermoFisher Scientific | 269-169400 | discontinued model, newer models available |