Name: using deuterium oxide as a non-invasive, non-lethal tool for assessing body composition and water consumption in mammals
Uploaded: 2020-02-20T12:00:00
Description: Watch this Scientific Journal Video about Using Deuterium Oxide as a Non-Invasive, Non-Lethal Tool for Assessing Body Composition and Water Consumption in Mammals at JoVE.com

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.

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
<ol>
	<li>Make a 50 mL stock solution of 9.0 g/L salinated D2O.
	<ol>
		<li>Weigh 450 mg of pharmaceutical grade NaCl and transfer all NaCl into a 100 mL, sterilized beake...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+serum+leptin+immunoreactivity+and+body+fat+mass+as+estimated+by+use+of+a+novel+gas-phase+Fourier+transform+infrared+spectroscopy+deuterium+dilution+method+in+cats.">Relationship between serum leptin immunoreactivity and body fat mass as estimated by use of a novel gas-phase Fourier transform infrared spectroscopy deuterium dilution method in cats.</a> American Journal of Veterinary Research. 61 (7), 796-801 (2000).</li><li>Moore, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Total+Body+Water+and+Solids+with+Isotopes.">Determination of Total Body Water and Solids with Isotopes.</a> Science. 104 (2694), 157-160 (1946).</li><li>Voigt, C., Cruz-Neto, A., Parsons, S., Kunz, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ecological and Behavioral Methods in the Study of Bats. , 621-645 (2009).</li><li>International Atomic Energy Agency. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Assessment of Body Composition and Total Energy Expenditure in Humans Using Stable Isotope Techniques. , (2009).</li><li>International Atomic Energy Agency. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to Body Composition Assessment Using the Deuterium Dilution Technique with Analysis of Saliva Samples by Fourier Transform Infrared Spectrometry. , (2011).</li><li>Shimamoto, H., Komiya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Turnover+of+Body+Water+as+an+Indicator+of+Health.">The Turnover of Body Water as an Indicator of Health.</a> Journal of Physiological Anthropology and Applied Human Science. 19 (5), 207-212 (2000).</li></ol>

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 metho...

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&#39;s muscle and superficial fatty tissue3. However, BCS assessment relies upon the evaluator&#8212;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).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 micron non-pyrogenic disk filter</td>
			<td>Argos Technologies</td>
			<td>FN32S</td>
			<td>nylon, 30mm diameter, 0.22um, sterile</td>
		</tr>
		<tr>
			<td>1.5 mL conical microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1415-9701</td>
			<td>1.5 ml self-standing microcentrifuge tube, natural with blue cap</td>
		</tr>
		<tr>
			<td>10 mL sterile glass vial for injection</td>
			<td>Mountainside Medical Equipment</td>
			<td>MS-SEV10</td>
			<td>clear, sterile glass injection unit</td>
		</tr>
		<tr>
			<td>10 mL syringe</td>
			<td>Becton Dickinson</td>
			<td>305219</td>
			<td>sterile 10 mL syringe individually wrapped</td>
		</tr>
		<tr>
			<td>100 mL sterile glass vial for injection</td>
			<td>Mountainside Medical Equipment</td>
			<td>AL-SV10020</td>
			<td>clear, sterile glass injection unit</td>
		</tr>
		<tr>
			<td>20 gauge needle</td>
			<td>Exel</td>
			<td>26417</td>
			<td>needles hypodermic 20g x 1&#34; plastic hub (yellow) / regular bevel</td>
		</tr>
		<tr>
			<td>22 gauge needle</td>
			<td>Exel</td>
			<td>26411</td>
			<td>needles hypodermic 22g x 1&#34; plastic hub (black) / regular bevel</td>
		</tr>
		<tr>
			<td>deuterium oxide</td>
			<td>Sigma-Aldrich</td>
			<td>151882-25G</td>
			<td>99.9 atom % D</td>
		</tr>
		<tr>
			<td>isofluorane</td>
			<td>Vetone</td>
			<td>3060</td>
			<td>fluriso isoflurane, USP</td>
		</tr>
		<tr>
			<td>OMNIC Spectra Software</td>
			<td>ThermoFisher Scientific</td>
			<td>833-036200</td>
			<td>FT-IR standard software</td>
		</tr>
		<tr>
			<td>petroleum jelly</td>
			<td>Vaseline</td>
			<td>305212311006</td>
			<td>Vaseline, 100% pure petroleum jelly, original, skin protectant</td>
		</tr>
		<tr>
			<td>plastic capillary tubes</td>
			<td>Innovative Med Tech</td>
			<td>100050</td>
			<td>sodium heparin anticoagulant, 50 &#956;L capacity, 30 mm length</td>
		</tr>
		<tr>
			<td>Sealed liquid spectrophotometer SL-3 FTIR CAF2 Cell</td>
			<td>International Crystal Laboratory</td>
			<td>0005D-875</td>
			<td>0.05 mm Pathlength</td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>EMD Millipore</td>
			<td>1.37017</td>
			<td>suitable for biopharmaceutical production</td>
		</tr>
		<tr>
			<td>Thermo Electron Nicolet 380 FT-IR Spectrometer</td>
			<td>ThermoFisher Scientific</td>
			<td>269-169400</td>
			<td>discontinued model, newer models available</td>
		</tr>
	</tbody>
</table>

using deuterium oxide as a non-invasive, non-lethal tool for assessing body composition and water consumption in mammals

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).

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 ...

Watch this Scientific Journal Video about Using Deuterium Oxide as a Non-Invasive, Non-Lethal Tool for Assessing Body Composition and Water Consumption in Mammals at JoVE.com

Using Deuterium Oxide as a Non-Invasive, Non-Lethal Tool for Assessing Body Composition and Water Consumption in Mammals

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 ...

Using this protocol, deuterium oxide, or D2O, a nonradioactive, stable isotope of water, can be used to estimate body composition and water consumption. This technique is noninvasive and therefore can be used to assess body composition in endangered species, as well as in humans and domestic species. A unique application of the deuterium oxide dilution technique is the ability to measure the water consumption of free-ranging wildlife with high recapture rates or of socially housed animals.While anesthesia is not required, it may be easier for those unfamiliar with giving subcutaneous injections to sedate the animals before delivering the deuterium oxide. Demonstrating the procedure with Sarah Hooper and me will be Amanda Eshelman and Ashley Cowan, veterinary students, and Alicia Roistacher, a graduate student from my laboratory. To make a 50-milliliter stock solution of nine grams per liter salinated deuterium oxide, first weigh 450 milligrams of pharmaceutical-grade sodium chloride, and record the exact amount to four decimal places.Transfer all of the salt into a sterilized 100-milliliter beaker, and measure out 50 grams of greater than or equal to 99.8%deuterium oxide in a sterile, graduated cylinder. Record the exact amount of deuterium oxide to four decimal places, and transfer the deuterium oxide to the sterile beaker of sodium chloride. Next, attach a 20-gauge needle to a nonpyrogenic, sterile disk filter with submicron pores fitted with a 10-milliliter syringe barrel, and insert the needle into the septum of a sterile, empty, 100-milliliter vial.Attach a vacuum tube to a 22-gauge needle, and insert the needle into the septum of the 100-milliliter vial. Then load 10 milliliters of isosmotic strength sodium chloride into the syringe barrel, and slowly turn on the vacuum until the deuterium oxide solution begins to slowly filter into the vial. Continue pouring the deuterium oxide stock solution into the syringe barrel until the entire 50-milliliter volume has been filtered.After confirming a lack of response to toe pinch in an anesthetized bat, use an alcohol prep pad to clean the uropatagium over the interfemoral vein, and allow the disinfected tail membrane to dry. Apply a thin layer of petroleum jelly over the interfemoral vein, and use a 29-gauge needle to puncture the dorsal portion of the interfemoral vein. Then use plastic sodium heparin capillary tubes to collect 100 microliters of blood.To ensure an adequate mixing of the whole blood with the sodium heparin, gently roll each tube after the blood collection before labeling. After determining the mass of deuterium oxide in grams to inject, load an insulin syringe equipped with a 29-gauge needle with the appropriate volume of deuterium oxide. Weigh the deuterium-loaded insulin syringe and needle, recording the weight to four decimal places, and inject the entire volume of deuterium oxide subcutaneously over the dorsal hip region of the anesthetized bat.We recommend using a precision scale with an antistatic draft shield and to make sure to pull back on the plunger to ensure negative pressure prior to injecting. Immediately after the injection, weigh the now-empty insulin syringe and needle, and record the weight to four decimal places. Within 30 minutes post-blood collection, use a hematocrit centrifuge to spin down each capillary tube at 10, 000 times g for five minutes.Using sharp scissors, cut the plastic capillary tube between the whole blood and the plasma, and use a 200-microliter pipette to expel the plasma directly into a labeled, 500-microliter storage tube. After the equilibration period, collect and store another 100 microliters of blood from the interfemoral vein as just demonstrated. For FTIR spectrophotometry analysis, set the temperature of a sand bath to 60 degrees Celsius to facilitate distillation, and add 50 microliters of each plasma sample and standard into individual 1.5-milliliter conical microcentrifuge caps.Keeping the microcentrifuge cap upside down, screw the 1.5-milliliter conical microcentrifuge tube onto the cap, and place the inverted tube in contact with the sand in the sand bath for a minimum of 12 hours. The next morning, place a new, clean cap onto each tube, and pulse the microcentrifuge tubes for 10 seconds in a microcentrifuge. Install a liquid transmission cell into the FTIR spectrometer, and fill the cell with methanol.Connect the injection port, and slowly fill the cell with background water while carefully removing the methanol syringe to reduce the risk of air bubbles. Attach tubing to the output port to allow removal of the samples post-analysis, and open the FTIR spectrometer software. Set the parameters according to the table, and collect a background sample using the diluent, 0.22-micrometer-filtered, distilled water.Inject 40 microliters of a zero parts per million deuterium oxide standard, and record and save the spectra as a comma-separated values file. After injecting and saving the spectra of all of the standards to create a standard curve, inject 40 microliters of each distilled sample into the liquid transmission cell, and save the spectra. With a known equilibration time, the total body water, lean body mass, and body fat mass for wild-caught big brown bats and captive big brown bats can be determined.A negative body fat mass can be 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 masses, or having under three to 5%body fat as determined by dual x-ray absorptiometry. Here, a representative percentage of body fat determined by the deuterium oxide dilution technique compared to dual x-ray absorptiometry is shown. The two techniques are well correlated, with the body fat mass showing strong correlations between the body fat and body weight and with the deuterium oxide dilution technique not consistently over or underestimating the body fat mass.The deuterium oxide dilution method has been previously validated in cats, and the technique was adapted to allow measurement of the daily water consumption of socially housed cats during each dietary block of the experiment. Remember that this technique should be performed only on healthy animals and that you should accurately weigh, record, and deliver the full dose of deuterium oxide to each animal. A secondary method for determining body composition, such as complete carcass analysis or DEXA, can be completed to validate the deuterium oxide technique.This technique allowed us to identify a socially housed cat that was consuming abnormally high quantities of water without separating each cat to measure their water consumptions.

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