The incorporation of quantitative measures of energetics is important for understanding the aspects of basic biology in relation to the physiology of aging and disease in organisms. Assessing changes in energy balance, body composition, and activity over the life course is important in evaluating interventions for functional improvements reflecting improvements foundational, biological changes that occur during the aging process. Multiple methods for the assessment of energetics have been validated and are available through the COEC.

  • Indirect Calorimetry (Mice)

    Energy balance studies are conducted using the TSE Systems PhenoMaster/Labmaster animal monitoring system (Bad Homburg, Germany). This system allows us to collect data on oxygen consumption, carbon dioxide production, food consumption and locomotor activity in 8 animals in parallel during the same measurement period. The entire unit is housed in an environmental chamber (Powers Scientific), allowing precise control of ambient temperature and photoperiod. Data are obtained using the TSE software. Mice are acclimated to the metabolic cage for two days prior to all measurements. Measurements are then taken for 22 hours or longer depending on the experimental design. Energy expenditure is calculated using 1-minute samples every nine minutes. Total energy expenditure is determined by calculating the average hourly energy expenditure over 22 hrs and then multiplying by 24. Resting energy expenditure is calculated by averaging the three lowest 18 consecutive-minute periods of energy expenditure, with at least one hour between each period. Locomotor activity is determined with infrared sensor pairs arranged in a grid pattern for horizontal (x, y level) activity. Movement is monitored continuously and reported as total counts every 9 minutes and expressed as counts/24 hrs. Food intake is monitored using hanging baskets attached to force transducers. Data on food intake is acquired every 9 minutes, and hence meal size, duration, and timing can be acquired. An automated food access system can be programmed to restrict access to food by time of day, duration of feeding, or by the amount consumed. Cages can contain a running wheel (n=8), which allows for exercise energy expenditure to be measured. The wheel can be always available, always locked, or controlled to allow only a certain time for running, or a specific distance run. A suspended tube in the cage is connected to a force transducer, which allows for measurement of body weight whenever the mouse enters the tube in its cage (n=8).

  • Indirect Calorimetry (Fish)

    The Loligo® respirometry system (Viborg, Denmark) measures resting oxygen consumption rates in aquatic organisms using intermittent flow respirometry. The system uses a fiber optic oxygen sensor, and data is collected and analyzed using AutorespTM data acquisition software. Oxygen consumption rates in up to four individuals can be measured simultaneously. The system is currently being optimized to measure individual oxygen consumption rates in fish ranging from early juvenile to adult under controlled temperature conditions. However, it can be adapted to measure oxygen consumption rates in a variety of aquatic organisms in either fresh or salt water. The system is currently being adapted to measure oxygen consumption rates in terrestrial organisms, specifically Drosophila melanogaster, and services should be available in the near future through the COEC.

  • Temperature/Activity

    The E-Mitter transponder/receiver system is used to measure core body temperature and consists of battery-free transponders that are implanted into the animal’s abdominal cavity and linked to a receiver base on which that the cage rests. The transponders transmit information on core body temperature via the base to a computer. Locomotor activity is assessed as the mouse moves over the top of the receiver. Thus activity and body temperatures can be measured continuously while the animal is in its home cage.

  • Wheel Running Cages

    Spontaneous exercise can be measured during or independently of indirect calorimetry using running wheels. Individual cage wheel use is recorded continuously for assessments of activity including total distance, speed and time of day.

  • Forced Exercise Wheels

    For investigators interested in the effect of forced exercise, the COEC has a system of running wheels on a rolling bed, for which the speed and time of the exercise can be determined and changed by the investigator.

  • Bomb Calorimetry

    Digestive efficiency can be assessed by measuring food intake and weight of feces produced and then measuring the energy content of samples of food and feces using a bomb calorimeter. By measuring the weight of food consumed and the energy content of that food, energy intake can be calculated. The weight and energy content of the feces can be measured to give an estimate of fecal energy lost, and together these give an indication of how much energy the animals are absorbing from the food.

  • Body Composition Analysis

    Body composition can be determined at the whole animal and/or tissue level using a variety of techniques. Which particular method would be best for your study design and organism can be determined in consultation with the Organismal Core leadership.

  • Chemical Carcass Analysis

    Chemical carcass analysis sets the benchmark for determination of body composition and the Organismal Core still uses this method to validate new instruments and techniques. It is also useful for animals (and derived tissues) that have been euthanized and frozen. The carcasses/samples are dried (for water content), fat is extracted, and combusted to quantify ash content. Common models include mice, rats and fish. For other species, please contact the Organismal Core.

  • Dual energy X-ray absorptiometry (DXA)

    DXA uses two different X-ray intensities to measure fat, soft-lean tissue, and bone in living animals. Animals are anesthetized (Isoflurane) and imaged with the front and back legs extended away from the body. The Organismal Core DXA machines can measure small (12~50g) and larger (250g-120kg) animals. A typical scan takes approximately five minutes.

  • Quantitative magnetic resonance (QMR)

    QMR is used to measure water content, fat, and lean mass in vivo with no need for anesthesia. The core routinely utilizes QMR to measure body composition across species (organisms/tissues) ranging in size from groups of 10 fruit flies, tissue samples (1g-10g), mice (15~80g), and rats (up to 900g). Scans take approximately two minutes for mice and rats, up to nine minutes for fruit flies. QMR measures with other species (e.g. aquatic, reptilian) may also be possible – please contact the Organismal Core for more information.

Healthspan Assessments

The Core is capable of assessing short-term physical activity measurements and cognition (memory and anxiety) using EthoVision® XT, a system for automated tracking and analysis of animal activity and behavior suited for everything from straightforward open-field tests to high-throughput research to sophisticated protocols including external equipment control.

EthoVision XT can be used to track any animal in any kind of test set-up. A basic EthoVision XT set-up allows you to automate standard behavioral tests such as the water maze, plus maze, and open-field test. In addition, EthoVision XT is easily used to automate high-throughput experiments, such as monitoring zebrafish larvae activity in 96-well plates. It is the industry standard with more than 7,500 peer-reviewed publications and is used at more than 2,000 sites worldwide.

The added benefit of this system is that we may use it to help local and external investigators add healthspan measures to their portfolio of assessments. This is because the Core is able to guide investigators in how to design and set up behavioral “arenas” to assess their healthspan measure of interest, in their local environment, and video record the behaving animals. Then, the Core will receive those videos and run them through the sophisticated Ethovision XT system. Thus, investigators without access to this state-of-the-art equipment or without knowledge of designing and performing behavioral assays may benefit from the expertise of this Core, even when located across the country.

In addition, we may assist locally with rotarod, grip strength, and exercise training, EchoMRI for body composition as well as various intervention methodologies (food, water, injection, or gavage delivery).

  • Drosophila lifespan studies

    We will conduct lifespan studies on capacity of a drug or genetic intervention to extend Drosophila lifespan.

    Drug: Compounds will be formulated into fly food at a range of doses (we recommend a dosage curve of 4 doses). Flies will be fed the drug until natural death. Animals will be transferred to new food and survival scored every 2-3 days (MWF). Evaluations may be performed in W1118 flies, Canton-S flies, Oregon-R flies, males, mated females, virgin females. We will evaluate 200-250 flies per condition. Charges will vary by number of conditions.

    Genetic: Gene overexpression or knockdown studies may be performed. This can either be performed by lines sent to us or we can assist in line selection.

    Negative geotaxis:
    Assessment of locomotor function. Flies will be transferred to a glass cylinder and allowed to acclimate for 10 minutes. The cylinder will be gently tapped to move all flies to the bottom of the chamber. The period of time until 40% of the flies has crossed an arbitrary mark placed at 12.5 cm will be recorded. Each vial will be evaluated five times and values averaged between trials.

    Spontaneous activity:
    Assessment of locomotor function. Spontaneous activity is monitored using a Trikinetic activity monitor. Activity is recorded for 48 hr in a humidified 24°C incubator with either a 12:12-hour light:dark cycle or constant dark as preferred. Flies will be allowed to acclimate for 8 hours prior to data collection.

    Olfactory Aversion Training
    Measure of learning and memory:

    Animals are exposed (via an air pump) in alternation to two neutral odors (3-octanol and 4-methylcyclohexanol, prepared as a 1/10 dilution in mineral oil) for 5 minutes under low red-light and a 100V 60Hz shock applied during exposure to one of the two odors. The odor associated with the electric shock is alternated between vials. After three training rounds per odor, animals are given one hour to recover then placed in a T-maze (Celexplorer labs) with opposing odors from either side. Flies are allowed two minutes to explore the maze after which the maze sections are sealed and the number of flies in each chamber scored. Results are evaluated by chi-square analysis.

    Smurf assay
    Measure of gut integrity:

    Dyed SY10 medium is prepared by addition of Blue Dye #1 (FD&C) at a concentration of 2.5% (wt/vol). Flies are maintained on dyed medium for 9 hours. A fly is counted as a Smurf when dye coloration could be observed outside of the digestive tract.

    Drosophila Lifespan Costs: (varies by experiment design, please discuss)

    Supplies and consumables for lifespan studies come to $0.10 per vial per day. A typical experiment of 40 vials run for 70-100 days

    Oxidative stress resistance:
    Flies are transferred onto vials containing half a Kimwipe soaked in 1mL of 5% sucrose and 10mM paraquat (Sigma). Every 8 hour following initiation of the challenge, flies are transferred to fresh vials containing the paraquat/sucrose medium, and dead flies are counted after each transfer.

    Heat stress resistance assays:
    Flies are exposed to heat (38°C) by sinking vials in a water bath. Numbers of dead flies were recorded every 30 min until all flies were dead.

COEC Energetics-Related Equipment List

  • Indirect Calorimetry/Metabolism Measurements
    • 8-cage TSE indirect calorimetry system with oxygen and carbon dioxide analyzers, infra-red beam break activity system
    • 8-cage TSE indirect calorimetry system with oxygen and carbon dioxide analyzers, infra-red beam break activity system running wheels, and automated feeders
    • 16-cage acclimation system for the indirect calorimetry system
    • Environmental Variables and Energetics Assessment - 3 environmental chambers (Powers Scientific) to measure indirect calorimetry of mice at different temperatures and light cycles
  • Activity and Energetics Measures
    • 32 wheel running cages: 16 for rats, 16 for mice
    • 20 walking wheel forced exercise system (Lafayette Instruments®)
    • 12 E-mitter receiver bases; 12 PDT-4000 (rats) and 20 G2 (mice) implantable transponders to measure temperature and activity in home cages
    • 1 rectal probe for measurement of rectal body temperature in mice
  • Body Composition Measurements
    • 2 drying ovens
    • 12 Soxhlet fat extractors
    • 1 muffle furnace
    • 3 industrial blenders
    • 3 DXA systems to measure in vivo body composition in animals from 12~50g and 250g to 120kg
    • 2 QMR systems to measure in vivo body composition in mice and rats (or similar sized animals)
    • 1 bomb calorimeter for the measurement of the energy content of food and feces
  • Healthspan measurements
    • EthoVision® XT with two camera systems, for automated tracking and analysis of animal activity and behavior (cognition, anxiety, social interactions)
    • Rotorod (1 for mice, 1 for rats)—balance and endurance
    • 1 Grip Strength (for both mice and rats)—strength
    • 1 Treadmill (for mice and rats)—endurance and exercise protocols
    • 1 EchoMRI for both mice and rats—body composition

Organismal Core Fees

Fees for the Organismal Core services are:

  • Indirect calorimetry: $6.44/animal/day (minimum $19.32 for 2 day acclimation & 1 day measurement)
  • Indirect calorimetry (fish): $50/animal
  • Activity/body temperature measurements: $2/animal/day; $25/surgery for transponder implantation
  • Wheel running cages: $3.62/animal/day
  • Forced exercise wheels: $2/animal
  • Bomb calorimetry: $25.72/sample
  • MicroCT (uCT): $78.63/hour
  • Carcass analysis (mice): $34.30/animal
  • Carcass analysis (rats): $50/animal
  • Carcass analysis (fish): $15/sample
  • DXA small (for mice): $8.26/animal
  • DXA large (250g to 120kg): $12.50/animal
  • QMR (any size up to ~900g): $7.68/animal
  • Analyses of EthoVision data (remote or local):$20/hour
  • Other behavioral assays: $20/hour
  • Drosophila lifespan costs: (varies by experiment design, please discuss), Supplies and consumables for lifespan studies come to $0.10 per vial per day. A typical experiment of 40 vials run for 70-100 days
  • Negative geotaxis (climbing assay): $50 per run
  • Spontaneous activity (Using DAM activity monitor, 24 vial capacity): $50 per 3 day run in 24hr light dark cycled incubator
  • Gut integrity (smurf assay): $20 per run
  • Olfaction Aversion Training (learning and memory): $100 per run
  • Oxidative stress/heat stress resistance (Survival assay to paraquat feeding or heat stress): $100 per run