BIO 3A Lab: Animal Metabolism Page 1 of 7 BIO 3A LABORATORY Animal Metabolism Objectives • Determine the raw oxygen consumption of mice at two different ambient temperatures. • Calculate weight specific metabolic rate adjusted to STPD for a homeotherm • Measure metabolic rate in a heterotherm, the goldfish, at different ambient temperatures Introduction In the reactions known as cellular respiration, oxygen is always reduced; it is the ultimate proton (H+) acceptor in this process: Sometimes this occurs almost immediately (aerobic metabolism); occasionally it is delayed (anaerobic metabolism). Temperature can have a profound effect on the rate of metabolic reactions. In general, metabolic reactions are governed by the Q10 rule. As temperature is increased by 10 degrees C the rate of the reaction will double. Your earlier labs involving proteins showed us the temperature sensitivity of enzyme-mediated reactions. However, in animals with variable body temperatures (heterotherms), several forms of an enzyme may exist (isoforms or isozymes). Each of these forms may demonstrate maximum activity at a different temperature. Thus, the organism may be able to survive over a range of ambient (and body) temperatures. Another group of animals puts a lot of energy (literally) into maintaining a constant body temperature. By doing this, they can minimize the number of forms of an enzyme required; in addition, if these animals maintain a temperature slightly below the critical maximum (point of protein denaturation) they can maximize the reaction rates (Q10 rule). These organisms are called homeotherms. Indirect measurement of metabolism in a heterotherm Goldfish are heterothermic ectotherms. Their body temperature is virtually identical to the water in which they live. Small variations in Tb can result from the exploitation of the variable microhabitat in which they live. For instance, the area of water nearer the surface may be slightly warmer; water under plant leaves may be cooler. However, in general, their Tb will vary through the daily cycle. The temperature-related metabolic response of most ectotherms is linear over a range of reasonable environmental temperatures. C6H12O6 + 6O2 → 6CO2 + 6H2O (1) Figure 1. Heterotherm metabolism vs. temperatureBIO 3A Lab: Animal Metabolism Page 2 of 7 At extremely cold temperatures, ice crystals form, causing fatal cell rupture. At higher temperature a critical maximum temperature is generally reached, at which enzyme activities, and reaction rates, are maximized. Beyond this temperature protein denaturation results in decreased activity and ultimately death. Procedure A. Indirect respirometry of goldfish metabolism The gill structures of most fishes are cover by an operculum. The operculum works in concert with the mouth and buccal cavity in moving water over the gills. The mouth opens, the buccal cavity expands, the mouth closes, the operculum opens and the buccal cavity contracts. This forces water over gills where oxygen and carbon dioxide are exchanged. If the volume of water is constant during each of these cycles, then the rate of opercular pumping is proportional to the oxygen demand. Thus one could indirectly measure metabolic rate as opercular pumping rate. In this experiment you will measure the opercular pumping rate of goldfish at three different temperatures. 1. Place a goldfish into approximately 200 mL of water in a 250 mL beaker. Place the beaker in the ice bath until the temperature has reach approximately 3 to 4 ˚C. 2. Remove the beaker gently and place it in a good viewing area. Count the opercular pumping for 2 min (120 sec). Repeat this measurement two more times. Record your data in Table 1. 3. Place the beaker into a room temperature bath. When the temperature has reach the ambient, let the system equilibrate for ten minutes. 4. Remove the beaker gently and place it in a good viewing area. Count the opercular pumping for 2 min (120 sec). Repeat this measurement two more times. Record your data in Table 1. 5. Place the beaker into the 30 ˚ C bath. When the temperature has reach bath temperature, let the system equilibrate for ten minutes. 6. Remove the beaker gently and place it in a good viewing area. Count the opercular pumping for 2 min (120 sec). Repeat this measurement two more times. Record your data in Table 1. 7. Using Excel, create a data table, in this table you should take the average of the pumping rate at each temperature. Create a graph of opercular pumping rate vs. ambient temperature. Is there a significant difference between the average opercular pumping rate at the low temperature and the high temperature? (hint: use a t-test!) Using the whole class data set (download this) for the goldfish experiment, is there a significant difference between the average opercular pumping rate at the low temperature and the high temperature? Measurement of oxygen consumption in a homeotherm Mice are homeothermic endotherms. They maintain relatively constant body temperature (Tb) throughout the day. Thus, it should be expected that mice would increase their metabolic rate to maintain constant body temperature if the ambient temperature (Ta) begins to fall. Figure 2. Operculum of aBIO 3A Lab: Animal Metabolism Page 3 of 7 In Figure 3 you can see the temperature-related metabolic response seen in most endotherms. TNZ refers to the thermoneutral zone, or the range of temperatures over which the animal's metabolic rate remains fairly constant. At these temperatures, the animal's metabolic rate is as low as it can be during the waking state. The lower critical temperature (LCT) is the point where metabolic rate increases to assist in maintaining body temperature in the face of the decreasing temperature. UCT, or upper critical temperature, is the point where energy is utilized to loose excess heat. The slope of the oxygen consumption rates above and below the critical temperatures, and the width of the TNZ are related to the type of animal, its size and its evolutionary history. For instance, the arctic fox, an animal well adapted for the life at cold temperatures, has UCT of only 0 °C. Even more startling is their TNZ, which extends far below -10 °C. Humans, on the other hand, evolved in a warm climate, witnessed by their narrow TNZ, high