BIOPAC Systems, Inc., 42 Aero Camino, Goleta, CA 93117 Ph: 805/685-0066 * Fax: 805/685-0067 * Web: http://www.biopac.com/ * E-mail: info@biopac.com BSL PRO Lesson #A07 2004 BIOPAC Systems, Inc. Updated 11-04-08 Fish Respiration and Q 10 Lesson provided by Dr. Peter Bushnell, Professor of Biology, Indiana University, South Bend Oxygen consumption in goldfish, recorded with the Biopac Student Lab and Dissolved O 2 Probe Transducer (SS69L); shown with raw and smoothed data overlapped. In this lesson, the Q 10 principle is demonstrated by measuring the metabolic rate of goldfish at two different temperatures: 22 o C (acclimation temperature) and 32 o C (acute exposure temperature). The fish are placed in a sealed metabolic chamber with a known amount of oxygen that will decline as the fish consumes it over time (~30 min). Temperature of the water in the chamber is controlled using a water bath. Changes in oxygen levels are recorded using the BIOPAC MP30 System for later data analysis. OBJECTIVES
1. Demonstrate the Q 10 principle by measuring the metabolic rate of goldfish at two different temperatures: 22 o C (acclimation temperature) and 32 o C (acute exposure temperature). Monitor the decline in a known amount of oxygen as the fish consumes it over time (~30 min.) in a sealed chamber of water at each temperature, using the BIOPAC Dissolved Oxygen Probe Transducer (SS69L). Record the change using the BIOPAC MP30 System. Measure the change in the partial pressure of oxygen (PO 2 ) and use the solubility coefficient to calculate the change in content. 2. Calculate the oxygen consumption of the fish at the two different temperatures. 3. Calculate the Q 10 of fish acclimated to 22 o C. BACKGROUND Poikilothermic animals are animals in which body temperature fluctuates more or less with the ambient temperature. Small fishes are poikilothermic because of the high heat capacity and heat conductance of water and the fishes' need to pass relatively large quantities of water over the gills in order to obtain oxygen. These factors prevent the behavioral thermoregulation observed in many terrestrial ectotherms. Enzyme activity is strongly dependent on temperature. In fact, a 10 o C change in physiological temperature range generally causes a 2-3 fold change in the catalytic rate of an enzyme. This principle applies to higher order processes as well, including metabolic rate, locomotion, and growth. A few processes, such as the biological clock, are insensitive to temperature. The factor by which the rate of a given process increases with a 10 o C increase in temperature is called the Q 10. It is calculated using the Van t Hoff equation: Q 10 = (k 2 /k 1 ) 10/(t 2 - t 1) where k 1 and k 2 are rates of reaction at temperatures t 1 and t 2, respectively. If the temperature range is 10 o C, this equation simplifies to: Q 10 = MR (t +10) /MR t The Q 10 depends on the particular temperature range. For example, the rate of a particular reaction may change more between 5 o C and 15 o C than it does between 10 o C and 20 o C. The temperature range must therefore be specified when giving a Q 10. Poikilothermic animals are able to respond to prolonged changes in temperature by up-regulating or downregulating enzymes in order to keep physiological processes operating at more or less constant rates at different temperatures. For example, imagine a group of goldfish that are living in water of 20 o C. All the goldfish have approximately the same metabolic rate. Half the fish are then moved to water that is 10 o C. The fish in the colder water seem much more sluggish and, at first, have lower metabolic rates than those remaining in the warmer water.
Within a few days, however, the metabolic rates of these cold water fish are approximately the same as their warmwater brethren. Fish may employ two strategies for such adjustments. Tetraploid fishes such as trout express different isoforms of an enzyme with different temperature-activity profiles. Such fish respond to changes in temperature in part by activating different genes. Most fish, however, are diploid and simply change the amount of an enzyme that is expressed in order to compensate for changes in temperature. In other words, when it gets colder and enzymes are less active, they simply raise the concentrations of enzymes. When it warms up, the concentrations of enzymes are decreased. Both of these strategies are long-term solutions -- it takes several days for the levels of an enzyme to increase when gene transcription is activated. There are a number of relatively easy ways to indirectly estimate the metabolic rate. The most commonly used approaches depend on the stoichiometric relationship between oxygen consumption, carbon dioxide production, and ATP generation, which is summarized by the equation for cellular respiration: 6O 2 + 6C 6 H 12 O 6 > 6 CO 2 + 6 H 2 O + 36 ATP Depending on the organism involved, one typically measures either oxygen consumption or carbon dioxide production. The high solubility of C0 2 in water makes it technically more difficult to measure C0 2 easily, so the metabolic rates of aquatic organisms are generally monitored by following the rate at which oxygen is consumed (mg or ml or Mol of O 2 /body weight/unit time). Metabolic rates measured in this manner are then multiplied by a constant to arrive at an estimate of energy expenditure (Watts, Joules/min, Cal/min). The temperature-metabolic rate relationship is illustrated in the graph above, which shows the change in oxygen consumption in goldfish exposed to ~5 o C steps in temperature change. The oxygen consumption at 25 o C is 26 times higher than at 0 o C. It is also clear that the change in VO 2 over a given change in temperature is not constant, but increases progressively with temperature. (Note: Stress also increases VO 2. Therefore, when handling the fish or doing anything associated with them, do it slowly and gently so they do not become terrified.) Oxygen Content versus Oxygen Partial Pressure (PO 2 Oxygen content and partial pressure are related through Henry s law, which states that the quantity of gas in a
solution (at equilibrium) equals αp, where P is the partial pressure of gas and α is the solubility coefficient, which varies with the type of gas, the temperature, and the liquid in which it is dissolved but is constant for a given gas at a given temperature in a given liquid. An increase in water temperature and/or salinity decreases its solubility for oxygen. In this lesson, the change in the partial pressure of oxygen (PO 2 ) is measured and the solubility coefficient is used to calculate the change in oxygen content. The Oxygen Electrode The oxygen content of the water is measured using a BIOPAC SS69L Dissolved Oxygen Probe. It is the most common type of oxygen electrode (probe) in use today, a polarographic oxygen electrode. The oxygen probe consists of an inner electrode that has an anode at the tip and a cathode band in the middle. The inner electrode screws into a plastic jacket filled with an electrolyte solution which electronically bridges the anode and cathode and allows a minute current to flow between the two. The electrode jacket is tipped with a thin, oxygen-permeable, Teflon membrane that isolates the electrode and electrolyte solution from the water sample. As oxygen diffuses across the membrane, the resistance of the electrolyte changes and the current flow between anode and cathode is altered in direct proportion to the oxygen flux across the membrane. A meter connected to the electrode provides a small polarizing voltage to the probe, measures the change in current with oxygen content of the sample, and displays it, or in this case, displays the level of O 2 via the Biopac Student Lab PRO software. Like all transducers, oxygen probes must be calibrated against known standards. (See calibration procedures below.) Two solutions are used for calibrations: one solution of water with no oxygen (sodium bisulfite), and a water sample with a known amount of oxygen (air-saturated water). Since metabolism is measured at two different temperatures, the electrode must be calibrated at each temperature. EQUIPMENT BIOPAC MP30 BIOPAC Dissolved Oxygen Probe Transducer (BIOPAC SS69L) or your existing Vernier O 2 probe with a BSL Interface (BSL-TCI16) Respirometer Air pump and air stones Water Bath Magnetic stirring plate Electrolyte Solution Zero % O 2 Solution (~20-30 mg sodium bisulfite / ml water) Goldfish acclimated to room temperature (~22 o C), the bigger the better Data Analysis Q&A Worksheet (click to download) SETUP Metabolic Chamber Setup 1. Set up the metabolic chamber (respirometer) and water bath. To measure metabolism, the fish are placed inside a small, sealed respirometer which sits in a larger, constant-temperature, water bath. To insure homogeneous temperature and oxygen distribution in the respirometer, the water bath and respirometer are placed on top of a magnetic stirring plate. A small magnetic stir bar, placed in the bottom of the respirometer, is then spun at a speed fast enough to mix the
water, but not overly exercise or stress the fish (i.e., no whirpools!). A false bottom made from plastic grate mounted on rubber stopper "feet" keeps the fish from being beaten-up by the stir bar. The setup described in this lesson utilizes a plastic container with two holes drilled into the lid. The oxygen probe, mounted in a rubber stopper, is placed into the larger hole. The second, smaller hole, is used as an access port in which to place a small air line to aerate the chamber between metabolism measurements and to vent air bubbles. Metabolic Chamber Setup: There are several ways to set up a metabolic chamber. One sample is shown below: The outer water bath is opaque to limit visual disturbance of the fish. The small clear window, usually covered with paper towel, allows observers to "peek" in at the fish, look for bubbles, and check the stirring bar. For assistance in preparing your lab for this lesson, contact BIOPAC. 2. Calculate the volume of the metabolic chamber. 3. Use a graduated cylinder to measure the chamber volume with the lid on, and any grate or stirring bar that may be in place. 4. Measure the volume three times and calculate the average. 5. Note the volume of the respirometer for later use on the Data Analysis Q&A Worksheet, item 3. 6. Put the fish into the metabolic chamber. a. Fill the respirometer about ½ full with tank water. b. Gently net a fish from the aquarium tank (the bigger the fish, the better). c. Place the fish in the respirometer and place the respirometer into the water bath.
If you are going to be using the water bath as a source of air-saturated water for calibrating purposes, make sure it is well aerated. d. Finish filling the respirometer with water from the bath. e. Replace the lid and center the respirometer on the stirring plate so the stir bar spins freely (if the chamber floats you may have to place a small weight on the lid to hold it down). f. Allow at least 30-60 minutes for the fish to recover from handling stress and its metabolic rate to return to normal. The longer you can wait, the closer the metabolism will be to standard metabolic rate. 7. Aerate the water in the respirometer and check stir bar. a. Place a small airline in the chamber and aerate the water during the acclimation period. b. Set the stirring bar to spin at a slow speed. Remember, you are trying to let the fish calm down so its metabolism returns to something approaching "resting". Overly vigorous aeration and/or stirring are going to stress the fish and increase its metabolism. Solutions In order to calibrate the oxygen electrode, you need to have two solutions: 1. Zero solution with no oxygen in it (zero solution, PO 2 = 0 mmhg) Zero solution should be made fresh every day by dissolving the Sodium Bisulfite in the appropriate volume of water. 2. Air-saturated solution that is equilibrated with atmospheric oxygen (air-saturated, PO 2 = whatever you calculated it to be in Step 3). Air-saturated water can be produced by vigorously bubbling the water bath itself with an air-stone, or bubbling a small beaker full of water in the water bath (the latter insures that the water is truly airsaturated and at the measurement temperature). In either case, the water should be bubbled for at least 30 minutes to insure full air-saturation. MP30 Hardware Setup 1. Connect the SS69L to CH 1 on the MP30, as shown here: Probe Setup 1. First use: a. Unscrew the probe tip, fill it with electrolyte solution, and screw the tip back on.
b. Remove the protective cap and immerse the probe in water for 2-3 hours before first use (this will charge the probe.) 2. Launch the BSL PRO software and open the Fish Q 10 template to establish the required software settings. Choose File > Open > Choose Files of type: Graph Template (*GTL) > File name: "a07.gtl" 3. Wait about 10 minutes as a probe "warm up" period. Note: The warm up period must be repeated any time the probe is disconnected from the MP30 unit. Hints for minimizing measurement error: Allow a sufficient warm up period before use. Ten minutes should suffice. Since the probe itself consumes oxygen, make sure that water is always moving past the probe tip whenever you are measuring or calibrating. It is important that no air bubbles form on the tip of the probe. Allow the probe to recharge for 1-2 hours if you change the electrolyte solution or the membrane cap. Keep the membrane moist while the probe is connected to the MP30. Do not drop, shake, or excessively impact the probe. Calibration and Calculations 1. Download and print the Data Analysis Q&A Worksheet so you can make notations as required for analysis. 2. Enter the following values into the appropriate spaces in your worksheet: a. Barometric pressure (mmhg) b. Temperature (oc) at which you are measuring fish metabolism (i.e. temperature of your water bath) c. Vapor pressure of water at the water bath temperature (see instructor for table of values). 3. Use the above values to calculate PO 2 and enter the result in your worksheet. You should get a number somewhere in the range of 150-160 mmhg. PO 2 = [Barometric pressure - Vapor pressure at bath temp] x.2095 4. In BSL PRO, click MP30>Setup Channels. 5. In the Setup Channels dialog, activate the three check boxes next to the "CH1" label by clicking each of them.
6. In that same row, click the "View/Change Parameters" wrench icon to generate the Input Channel Parameters dialog. 7. Click the "Scaling" button to generate the Change Scaling Parameters dialog. 8. In the Change Scaling Parameters dialog, label the units "mmhg" for millimeters of mercury or "torr" (equal measures). 9. Set Cal 1. a. Immerse the probe in Zero % O 2 solution and wait two minutes. b. Stir the probe a little and then click "Cal 1." Calibration values should fall in the range of 0.2-0.5 V. If your values vary, move the probe to release a possible bubble on the tip.
Keep the liquid moving across the membrane surface to prevent air bubbles. c. Set the "Cal 1" Scale value to 0. d. Rinse the probe tip thoroughly in fresh water. 10. Set Cal 2. a. Immerse the probe tip in air-saturated water drawn from the water bath or water beaker at the equilibration temperature. b. Wait one minute, then stir the probe and click "Cal 2." c. Set the "Cal 2" Scale Value to the PO 2 you calculated for the day (150-160 mmhg). d. Click "OK" and close out of any remaining dialog boxes. 11. Insert the probe into the large hole in the respirometer and allow it to equilibrate to the water bath temperature as the fish is acclimating to its new surroundings. RECORDING To calculate the Q 10 you must measure oxygen consumption / metabolic rate at both the acclimation temperature and the acute exposure temperature. Metabolic rate is determined by recording the fall in PO 2 that occurs as the fish consumes oxygen in the respirometer. It is also good practice to record data by hand, so you should also note the PO 2 in your lab book at regular intervals (every minute, perhaps). If all goes well, a graph of PO 2 over time produces a straight line relationship with a negative slope (change in PO 2 /time). A linear regression of PO 2 over time generates a value for this rate of decline (slope) which is then used to calculate metabolic rate. Measure metabolic rate (fall in PO 2 ) at acclimation temperature 1. Prepare the equipment for measuring. a. Take the air line out of the respirometer. b. Carefully (and quietly) check to make sure there are no bubbles trapped under the lid of the respirometer. If you see bubbles under the lid, gently tilt the chamber until the bubbles escape from the small vent hole, and then seal it with a rubber stopper. c. Check the stirring bar to make sure it is slowly spinning and mixing the water. 2. Record the fall in PO 2 with the BIOPAC MP30.
a. If you are not already monitoring the PO 2 in the chamber, press the "Start" button in the lower right portion of the BSL PRO graph window. If this is your second metabolic rate measurement, make sure you are not overwriting data you want to save! b. Continue taking readings until PO 2 falls by about 10 mmhg, or ~20-30 minutes has elapsed. Manually jot down PO 2 readings every few minutes to compare with data on the computer. As time progresses, the PO 2 should decline in a relatively linear manner with a noticeable slope. If not, consult with your instructor. c. Press "Stop" in the software window. 3. Prepare the equipment for a second measurement at the same temperature. a. Re-oxygenate the chamber. Take the small rubber stopper out and replace the small airline in the respirometer. b. If after 10 or 15 minutes the PO 2 of the chamber has not returned to approximately the same place the the first measurement cycle began, you might have a calibration error. Recalibrate the "top-end" of the calibration curve by placing the electrode in air-saturated water Follow Calibration procedure steps 9-10. 4. Measure the metabolic rate again at the same temperature. a. Follow these procedures for measuring metabolic rate steps until you have at least two measurements. If prior measurement reduced the PO 2 in the chamber significantly, do not begin a new measurement until the PO 2 in the chamber has risen above 145. Expose the fish to an acute temperature change An acute temperature change can be produced by either warming or cooling the water bath. For ease of calculation, a 10oC change is recommended, but not necessary. Increasing temperature of the water bath 1. Warm the water bath. a. Warm the water bath by carefully pour in small amounts of hot water into the water bath until you raise the water bath temperature by 10 o C. Do not add hot tap water to the tank as it chlorinated and will kill the fish. Always use dechlorinated water provided by the instructor. Remove equal amounts of water from the bath as you add more hot water. Make sure the bath is stirred and thoroughly mixed. Once desired temperature is achieved, monitor the temperature and add hot water as needed. 2. Once the water bath is warmed you must wait until the water in the respirometer also reaches the new temperature. a. Make sure that the chamber is aerated throughout the equilibration period. b. In order to speed up the process you can slowly and quietly replace some of the water in the respirometer by using a large syringe or turkey baster to squirt warm bath water into the respirometer. c. Use a thermometer to confirm full equilibration. d. Once the chamber water has reached the new equilibration temperature, wait ~15-30 minutes for the fish to calm down and oxygen electrode to equilibrate to the new temperature. Decreasing the temperature of the water bath
1. Cooling the water bath- There are two ways two cool the bath- add ice made from dechlorinated water, or add freezer packs placed inside ziplock bags (prevents accidental contamination of the water) 2. Monitor the temperature change with a thermometer and add or remove ice (packs) as needed. 3. Once your have reached the new equilibration temperature, equilibrate the respirometer water as outlined in #2 above. Measure the metabolic rate (fall in PO 2 ) at the acute exposure temperature 1. Recalibrate the electrode. a. Because temperature changes the solubility of water, as well as the response characteristics of the electrode, it must be recalibrated at the new temperature. Once again, it is usually only the "topend" (air-saturation) that must be checked. b. Follow calibration procedure in step #10 in the Calculation and Calibration section above. 2. Measure the metabolic rate at the new temperature. a. Measure twice, following the same procedure used above to measure metabolic rate at acclimation temperature, but this time at the new acute exposure temperature. Determine the mass of the fish After recording and determining metabolic rate, weigh the fish and return it to its home tank. To determine the mass of the fish: 1. Remove the electrode from the respirometer and empty about half the water out of the chamber (make sure there is enough water for the fish to be comfortable). 2. Carefully dry the outside of the respirometer, place it on the balance and and tare it to 0. 3. Remove the respirometer from the balance, remove the lid, and carefully dip net the fish out of the chamber. Try and make sure that all the water drips back into the respirometer. 4. After placing the fish back in its home tank, replace the lid on the respirometer and reweigh it. The balance reading will be the mass of the fish (why is the reading negative?) 5. Record the mass (in grams) on the Data Analysis Q&A Worksheet, item 5. DATA ANALYSIS Click to download the Data Analysis Q&A Worksheet to calculate metabolic rate. In order to calculate metabolic rate, first determine the slope of the slope of the line (decline in PO 2 over time) recorded by the BIOPAC MP30 System. To determine slope: 1. Export the BIOPAC data files as *.txt files. 2. Import them into Excel or QuatroPro. 3. Use the linear regression function to calculate slope and regression coefficient (r 2 ). See Calculation 8 of the Data Analysis Q&A Worksheet for further instructions on how to calculate slope.
Use slope and other calculations on the Data Analysis Q&A Worksheet to determine the metabolic rate and the Q 10 of 22 o C acclimated fish. GRAPH TEMPLATE SETTINGS The BSL PRO Graph Template file a07.gtl automatically establishes the software settings for this lesson. Click here to open a PDF of the graph template file settings; the settings are provided for reference only -- you simply open the template file and they are activated. Return to PRO Lessons Index