Tordoff3 project protocol

Diet preference: Survey of calcium and sodium intake, blood pH and calcium level, and bone and body composition data in 40 inbred strains of mice   (2002)

Tordoff MG, Bachmanov AA

See also: Tordoff3 animal documentation


Mice were assessed for:

AssayOrder and timing of testingEquipment
Ingestive behavior6-10 days after shippingSee description below
Blood calcium and pH~7 days after behavioral testing completed Ciba-Corning Calcium Analyzer (model 634)
Bone and body compositionat least 6 days after blood sample taken PIXImus II Mouse Densitometer (model 51601)


Assays

Behavioral Tests
Construction of drinking tubes and other experimental procedures are given in detail at www.monell.org/MMTPP. Each drinking tube consisted of a 25-ml plastic serological pipette with 0.2 ml gradations (Fisher cat no. 13-678-14B). This was connected to a 63.5-mm long stainless steel straight sipper tube (Unifab, Cat. No. US-171-25) with a 15-mm piece of silicon tubing (Cole Palmer, Cat. No. 06411-76). The top of the pipette was closed with a size 00 rubber stopper. Tubes were always given in pairs, with one containing deionized water and the other taste solution. The drinking tubes were placed to the (experimenter's) left of the food hopper. The spouts extended ~25 mm into the mouse cage and their tips were ~20 mm apart. Each spout had a tip with a 3.175-mm diameter hole from which the mice could lick fluids.

After 6-10 days habituation to laboratory conditions, the mice were tested with the following compounds in the order listed.
OrderTestSigmaConcentrationsDays
1water vs. water  2
2CaCl2C-38817.5, 25, 75 mM6
3Calcium lactate (CaLa)L-20007.5, 25, 75 mM6
4NaClS-962525, 75, 225 mM6
5Sodium lactate (NaLa)L-702225, 75, 225 mM6
 Total duration:  26


Compounds were purchased from Sigma-Aldrich, and were prepared in deionized water. The "water" drinking tube also contained deionized water. Each test was 48 h in duration and the positions of the two drinking tubes were switched after 24 h to control for side preferences. Intakes were measured to the nearest 0.1 ml at the end of each test. Extensive experience has shown that fluid spillage and evaporation is ~0.25 ml over 48 h with these procedures, and so these were ignored.

All measurements were made in the middle of the light period (late morning or early afternoon). Body weight was measured (to the nearest 0.1 g) at the beginning and end of the 24-day test series.

Data available include preference scores for three concentrations of each solution.

Notes: A few (94 out of 37,920) measurements were lost due to spilled or blocked drinking tubes, or other technical problems. When intake was compromised, the mouse was retested at the end of the series. The results of these retests are included in the submitted data set. Retested mice (per measurement) are disclosed at www.monell.org/MMTPP.

Analysis of blood ionized calcium and total plasma calcium
Approximately one week after the end of behavioral tests, a blood sample was taken from each mouse. The mouse was removed from its cage and restrained in a paper towel. The tip of its tail was cut with a scalpel and two samples of 25 - 30 microliters blood were gently milked into 60 microliter microhematocrit tubes treated with ammonium heparinate (Fisherbrand cat no. 22-362-566, Fisher Scientific). Using a stopwatch, the time was measured from cutting the mouse's tail until the first sample was collected. This served as a rough measure of the ease of handling and bleeding time of the mouse, and as a control for blood pH, which is time-sensitive. The first sample was immediately analyzed for ionized calcium and pH using a Ciba-Corning calcium analyzer (Model 634) kept in the vivarium. The second sample was held until samples from 6-8 mice were completed then spun in a microcentrifuge located in the corridor outside the animal room. These samples were then refrigerated until 24 or more samples were available. Then, the plasma was separated and analyzed for plasma total calcium using a colorimetric assay based on Sigma-Aldrich Calcium Kit #587-S but miniaturized to use 7.5 microliters of plasma in a 96-well plate format. Due to occasional technical problems, a few samples were lost, in which case the procedure was repeated the following week.

Data are available for bleeding time, blood ionized calcium, blood pH, and plasma total calcium. Blood ionized calcium was adjusted to pH 7.4 and is provided in the data set as a derived field.

Bone and Body Composition

At least 6 days after the tail blood sample was collected, each mouse was anesthetized with 70 mg/kg ketamine + 10 mg/kg xylazine (5 ml/kg) and as much blood as possible (400 - 1400 uL) removed by cardiac puncture. The dead mouse was then wrapped in aluminum foil and frozen at -80°C for several months.

Frozen carcasses were allowed to thaw at room temperature for ~1 h, weighed using a top-loading balance, and then analyzed using a PIXImus II mouse densitometer (#51601; GE Medical Systems Lunar, Madison, WI). Each carcass was laid out on a plastic tray in the prone position with the limbs held splayed apart with tape. The tail was displaced to the mouse's left so that it was within the X-ray field but did not overlap a limb. Because the cone-beam X- ray field is only 80 x 65 mm and thus too small to fit a whole adult mouse, the head of each mouse was excluded from analysis; it was either placed outside the X-ray field or masked using software.

The PIXImus uses low energy X-rays to produce high-resolution (0.18 x 0.18 mm pixel) images of the mouse. PIXImus 2 2.0 software included with the machine calculates bone mineral density and content, and body fat and lean tissue content. The PIXImus was calibrated daily using a plastic "mouse phantom" provided by the manufacturer.

Data are available for:

  • Age at death
  • Weight of exsanguinated, thawed carcass (supplemental data)
  • Bone mineral density (BMD)
  • Bone mineral content (BMC)
  • Bone area: Calculated bone area on X-ray (supplemental data)
  • Lean weight: Calculated weight of lean tissue
  • Fat weight: Calculated weight of fat tissue
  • Total weight: Lean weight + Fat weight
  • Percentage of carcass accounted for as fat
  • Percentage of carcass accounted for as lean
Notes:
BMC = BMD x bone area
% Fat = (Fat weight / Total weight) x 100
% Lean = (Lean weight / Total weight) x 100