Author Institution: Department of Physiology, The Ohio State University, Columbus 1

Angerer, Clifford A.

Calhoon, Thomas B.

The Ohio Journal of Science

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A VOLUMETER FOR RESPIRATION OF AQUATIC ANIMALS1THOMAS B. CALHOON2 AND CLIFFORD A. ANGERERDepartment of Physiology, The Ohio State University, Columbus 10The methods for measurement of respiration of small aquatic animals arerather limited. The Wrinkler titrimetric method (Birge and Juday, 1911; Alleeand Oesting, 1934) has proved not only laborious but also time-consuming forlong-term routine determinations. The respirometric method described herehas many advantages over it: (1) continuous oxygen consumption determinationscan be made for several hours without disturbing the experimental animals, (2)the respirometric technique involving less manipulations is accordingly subjectto less experimental error, (3) the technique gives more linear rates of oxygenconsumptions than does the Wrinkler technique, especially when these rates areof low order (fig. 2).STOPCOCK — .r\.CO,ADSORBER\ .IIu cKf ^ ^ T O MEASURINGI t 11 SYRINGES11?S \ \ lOee. RESERVOIRV \ / i BULBSRESPIROMETER / 2«». CAPILLARY . / INDICATORCHAMBER TUBE>COMPENSATORY. -CHAMBERFIGURE 1. The volumeter used for respirometric studies (see text for discussion).The method to be described here (Calhoon and Angerer, 1949) is based onthe same general principle as that employed by Peiss and Vennesland (1949) andVennesland (1951). It consists in making determinations of volume changesfor a closed system maintained at constant pressure. Any decrease in gaseouspressure (oxygen consumed) is compensated by a decrease in total volume. Thedecrease in volume is measured by an appropriate movement of pistons havingknown displacements.The main flask, the respiratory chamber (fig. 1), is of ca 500 cc capacity andcontains the animal whose oxygen consumption is to be determined. When aJThis investigation was supported by a grant-in-aid from the Comly-Coleman ResearchFund, College of Medicine, and from the Graduate School, The Ohio State University.2Present address: Department of Physiology, The Medical College of the State of SouthCarolina, Charleston 16.THE OHIO JOURNAL OF SCIENCE 53(4): 240, July, 1953.No. 4 VOLUMETER FOR RESPIRATION 241fish is placed in the chamber, 300 to 400 cc of water are added; when a frog, 25to 30 cc; and when a small terrestrial animal, no water is required. The upperwall of the respiratory chamber converges to form a neck which is closed by aground-glass stopper held- in place by means of helical springs. Through theneck of the chamber, the animal is placed into the respirometer. From the neckof this chamber emerge 3 side-arms: one wide-mouth arm contains the CO2 absorber(10% KOH solution); a second side-arm is a vent with stopcock for changing thegaseous armosphere within the respiratory chamber: a third side-arm consistsof a 4 mm glass tube which connects to a 10 cc reservoir bulb. On the lower surfaceof this bulb is attached a 2 mm capillary tube which leads to another 10 cc reservoirbulb connected, in turn, to a compensatory flask. The capillary tube has anindex mark etched on it. The compensatory flask contains a few cc of waterto assure equal vapor pressure in both the respiratory and compensatory chamberswhen at the temperature of the water bath. The sensitivity of the system isTIME IN MINUTESFIGURE 2. The rates in minutes (abscissa) of the oxygen consumption in cc of oxygen con-sumed/gin wet weight/hr, (ordinate) for the same goldfish as studied by the volumetric(O) and by the Winkler ( • ) techniques. Temperatures of 30.00 ± 0.01 °C and 25.4°C,respectively.directly related to the gaseous capacity of the compensatory chamber. There isplaced inside of the index-marked capillary tube a colored fluid. The proximal(relative to the respiratory chamber) air-fluid interface of this column of fluid ismade to coincide with the index mark at the time of obtaining each reading of thevolume of oxygen consumed. This assures the maintenance of a constant gaseouspressure in the respiratory chamber. For compactness the extended third side-armwhich carries the index fluid and compensatory flask encompasses the respiratorychamber, and is annealed to it in several places in order to gain rigidity.Inserted into the ground-glass stopper are 3 small-bore outlets arising froma common vent. To each outlet is attached a syringe of any capacity dependenton the volume of oxygen to be measured during the course of the experiment.For studies on frogs and goldfish, syringes of 1 cc (100 division/cc) and 2 of either242 THOMAS B. CALHOON AND CLIFFORD A. ANGERER Vol. LI 115 or 10 cc capacity were most satisfactory. For more precise readings a micro-syringe designed by Woodrow (1949) may be conceivably incorporated. Thesesyringes are connected by pressure tubing. As the O2 and CO2 are removed bythe organism and KOH solution, respectively, the difference in pressure betweenthe respiratory and compensatory chambers forces movement of the index fluidtowards the proximal reservior bulb. By appropriate movements of the pistonsof the syringes, the proximal air-fluid interface is returned to the index mark andthe volume of oxygen lost to the system is read directly.The respirorrieters were immersed in a thermostatically controlled water-bath(30.00 =•= 0.01° C), and were held in position by a rack which was originally adaptedto fit into the shaft designed to hold Warburg manometers. The respirometersmay be oscillated for varying distances and for varying frequencies characteristicof the shaking apparatus in question.Although the volumeters have been used successfully on aquatic animals,goldfish and frogs (Calhoon and Angerer, 1949, 1950a, 1950b, 1951; Murray andAngerer, 1950), there is evidence that they may be adapted to small terrestrial ani-mals providing that they are not cramped in the respiratory chamber. Studies weremade upon the effect of shaking the apparatus at various frequencies. It wasfound that when the volumeters were not shaken they gave identical rates foroxygen consumption as when they were shaken 40 oscillations/minute. However,the values obtained with 40 oscillations/minute were sometimes variable. Further-TABLE 1Comparison of the respirometric and the Winkler methodsMethodsWinklerRespirometerNo. of Fish1818°C. Temp.25.4030.00*Mean M.M.0.2110.282*Std.0.0.Dev.070083Std.00Error.016.019*The difference in mean metabolic rate (M.M.) is due to the difference in temperature,assuming a Qio value of 2.7.more, shaking at 80 oscillations/minute caused a three-fold increase in oxygenconsumption over that obtained without shaking. This was due to the highoscillatory rate stimulating the animal to excessive movements and thus causingan increase in oxygen consumption. That the rate of oxygen consumption withoutshaking is indentical to that obtained with the Winkler technique is probablydue to the movements of the animal being sufficient to mechanically mix thecontained gas. Therefore, the respirometers were not shaken during the courseof these experiments.To compare the accuracy of the volumetric to the Winkler technique, oxygenconsumption determinations were made by both methods on a group of 18 goldfish,Carassius auratus. All fish, weighing between 5 and 10 grams, were starved 12to 18 hours prior to use in order to render them into, as near as possible, a post-absorptive state. The results are presented in Table 1 as cc of O2 consumed/gmwet weight/hr. The mean value for oxygen consumption is substantiallyhigher for the volumetric than for the Winkler technique (0.282 =•= 0.019 and0.211 ± 0.016, respectively). However, when correction is made on the basisof their Qi0 values for the difference in temperature (30.00 =•= 0.01° C and 25.4°C, respectively) at which the two sets of data were obtained, it is found that themeans of the two sets of data are identical. Despite the difference in temperatureat which the two techniques were run, the standard error (S.E., table 1) of themean values for oxygen consumption is almost the same ( ± 0.003) in both cases.No. 4 VOLUMETER FOR RESPIRATION 243SUMMARYA direct reading volumeter, requiring no previous calibration has been devisedfor study of the oxygen consumption of small aquatic and amphibious animals.It is conceivably possible to extend its use to small terrestrial animals. A truemean value of 0.282 ± 0.019 cc O« consumed/gm weight/hr, at 30.00 ± 0.01°C was obtained for the common goldfish, Carassius auratus, by the volumetricrespirometer. This value compares favorably with a true mean value of 0.211=*= 0.016 for the same fish at 25.4° C using the Winkler technique. The volunteers,therefore, show degrees of accuracy similar to the Winkler technique. An advant-age in favor of the volumetric over the Winkler technique is found in the greaterlinearity of the points in the graph on,plotting the cc of oxygen consumed as afunction of time (fig. 2). Finally the volumetric respirometer reduces the laboriousand time-consuming manipulations inherent in the Winkler technique.LITERATURE CITEDAllee, W. C, and R. Oesting. 1934. A critical examination of Winkler's method for determiningdissolved oxygen in respiration studies with aquatic animals. Physiol. Zool., 7: 509-541.Birge, E. A., and C. Juday. 1911. The inland lakes of Wisconsin. Bull. Wise. Geol. and Nat.History Survey No. 22.Calhoon, T. B., and C. A. Angerer. 1949. The antagonistic action of adrenal cortical extracton the inhibitory metabolic effect of large doses of sesame oil. Anat. Rec. (Suppl.),105: 142.Calhoon, T. B., and C. A. Angerer. 1950a. A respirometer for the measurement of the oxygenconsumption of aquatic and amphibious animals. Anat. Rec. (Suppl.), 108: 3.Calhoon, T. B., and C. A. Angerer. 1950b. The effect of relatively large doses of sesame oilon oxygen consumption and the antagonistic action of adrenocortical extract. Endocrinol.,46: 327.Calhoon, T. B., and C. A. Angerer. 1951. Use of a new respirometer in studying the effects ofadrenocortical extract on total metabolism. Fed. Proc, 10: 23.Murray, Mary C, and C. A. Angerer. 1950. Adrenalectomy on oxygen consumption of frogs.Amer. Jour. Physiol., 163: 737.Peiss, C. N., and R. Vennesland. 1949. Comparison of a volumetric respirometer with theWarburg method in measurement of brain respiration. Fed. Proc, 8: 126.Vennesland, R. 1951. A volumetric microrespirometer for studies of tissue metabolism.Science, 114: 100.Woodrow, A. W. 1949. A convenient microsyringe. Science, 100: 142.

A Volumeter for Respiration of Aquatic Animals

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A Volumeter for Respiration of Aquatic Animals

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