J. Nutr. Sci. Vitaminol., 28, 35-39, 1982 Levels of CO2 in Arterial Blood of Carp under Carbon Dioxide Anesthesia Hisateru MITSUDA, Saburo UENO, Hiroshi MIZUNO, Tadashi UEDA, Hiromi FUJIKAWA, Tomoko NOHARA, and Chieko FUKADA1 Interdisciplinary Research Institute of Environmental Sciences, Kawaramachi Kojinguchi-agaru, Kamigyo, Kyoto 602, Japan (Received May 23, 1981) Summary The application of carbon dioxide anesthesia to fish has been studied in order to find a method of transport of live fish. In the continuation of this study, the levels of arterial PaO2, PaCO2 and ph were examined in carp anesthetized with carbon dioxide. In the initial stage of the anesthesia, both PaO2 and PaCO2 increased. In the following stage, PaCO2 gradually decreased. An increase or decrease in PaO2 seems to depend on the levels of PaCO2 during anesthesia. On the other hand, ECG (electrocardiogram) profiles of anesthetized carp indicate that a gradual increase in R-R interval is coupled with a gradual decline of the opercular rate. Key Words anesthesia, blood carbon dioxide, Cyprinus carpio, electro cardiogram The transport of live fish has become of importance because fish taste very good and contain proteins which are rich in essential amino acids and present a good amino acid profile for human requirements. For this purpose, safe techniques and methods for the transport and handling of live fish have been intensively investigated. One effective method is the use of anesthetics on fish to suppress swimming and metabolism. The use of carbon dioxide as an anesthetic is promising, because compared to conventional anesthetics, carbon dioxide gas is not expensive, and is absolutely safe for humans. The idea of the use of carbon dioxide came from a series of food preservation experiments (1) which we developed from earlier findings on the relationship between environmental change and catalase activity (2). Evidence that catalase activity depends on body temperature is particularly important for poikilotherms which are sensitive to changes in the environmental temperature; this may account for one aspect of hibernation. This understanding led to the underwater and 35
36 H. MITSUDA et al. underground storage of cereal grains (3). Other evidence that catalase activity is sensitive to carbon dioxide (4) is of particular interest because it implies the possibility that hibernation or a similar behavioral pattern can be induced by carbon dioxide. In fact, we previously reported (S) that the behavior of carp when carbon dioxide is introduced into the water is similar to the behavior produced by other anesthetics (6). Carbon dioxide is certainly not detrimental to carp. Since a high level of carbon dioxide is lethal, control of the concentration of carbon dioxide in the water is essential to safe anesthesia. It is thus necessary to know the levels of CO2 in the arterial blood of carp under carbon dioxide anesthesia. MATERIALS AND METHODS Adult carp, Cyprinus carpio, ranging from 500 to 700g in body weight, were reared in the manner previously reported (S). Prior to the experiment the carp were acclimatized in a glass aquarium (45 ~45 ~30 (height) cm). The action of the carp under anesthetic was examined when the CO2 concentration in the water was changed by bubbling equi-mixtures of CO2 and O2 gas at various flow rates into 30 liters of fresh water. An equi-mixture of carbon dioxide gas (1 liter/min) and oxygen gas (1 liter/min) was then selected, but the gas was stopped after 10-min bubbling in order to induce a moderate anesthetic action. During the experiments, the concentration of carbon dioxide dissolved in the water was measured with a carbon dioxide electrode (model 95-2 Orion). Partial pressures of carbon dioxide (PaCO2), oxygen (PaO2) and ph in the arterial blood of carp were measured with an IL 213 05 blood gas analyzer (Instrumentation Laboratory). The oxygen saturation was estimated from the PaO2 and ph. Electrocardiograms (ECG) were monitored during the experiments in the manner reported previously (S) with a Polygraph RM -6200 (Nihon Koden). RESULTS AND DISCUSSION When the aquarium was aerated with an equi-mixture of the CO2 and O2 gas at various flow rates, the concentration of carbon dioxide dissolved in the water was as shown in Fig. 1. The time to when the carp begin to become excited or when the carp begin to lie on their sides is shortened as the flow rate of the gas mixture increases. A curve connecting points marked by 'E' (or 'L') on CO2 (ppm)-time curves shows a hyperbola, suggesting that the basis for an anesthetic effect of CO2 is not the level of carbon dioxide concentration in water, but an apparently cumulative quantity of carbon dioxide which carp consume. This depends on both the concentration of carbon dioxide dissolved in the water and the time of exposure of carp to CO2, and therefore is closely related with the levels of carbon dioxide in blood. As seen in Fig. 1, aeration with the gas mixture of CO2 and O2 initially gives a linear increase in CO2 concentration, producing fatal effects on carp if the aeration is continued. Therefore, aeration with the gas mixture must be stopped at J. Nutr. Sci. Vitaminol.
BLOOD GAS OF CO2 ANESTHETIZED CARP 37 Fig. 1. Relationship between changes in CO2 concentration in water and anesthetic actions of carp when aeration with equi-mixtures of CO2 and O2 gas are introduced. ' E' indicates that carp begin to become excited, and 'L' indicates that carp begin to lie on their sides. Fig. 2. Relationship between changes in arterial blood ph, PaCO2, PaO2 and oxygen saturation of carp, when 10-min aeration with CO2 and O2 gas was introduced. Vol. 28, No. 1, 1982
38 H. MITSUDA et al. appropriate levels of carbon dioxide in water. The level of the carbon dioxide is usually maintained for a few hours, and is then gradually decreased. According to our experimental results, 10-min aeration with a gas mixture of CO2 (1 liter/min) and O2 (1 liter/min) leads to considerably longer duration of sedation. Figure 2 shows changes in arterial blood gas pressures, ph, and oxygen saturation of carp under the above experimental conditions. The ph curve decreased rapidly and then gradually. After about 60min ph reached a constant value of 6.72 }0.05. Both PaO2 and PaCO2 increased in the initial stage of the anesthesia. The increase in PaCO2 seems to accelerate the oxygen uptake, although it is known that the concentration of carbon dioxide is related closely to the effects of CO2 on fish hemoglobin, and therefore high PaCO2 levels reduce the affinity of hemoglobin (7). This observation suggests that oxygen-rich water is needed if carp are anesthetized with carbon dioxide. A drastic change in PaO2 was observed, usually after about 60min, in contrast with a gradual increase in PaCO2. The Fig. 3. ECG profiles under CO2 gas anesthesia of carp. (1), (2) and (3) show the P wave, QRS-complex, and deep respiration, respectively. (A) The CO2 and O2 gas mixture was introduced. (B) The carp lay on their sides at 5 min. (C) The complicated background profile disappeared at 9min. One RS wave is deficient in the middle of the profile. (D) P waves disappeared completely. In contrast, unidentified small negative waves which followed the RS waves 0.5 sec later were regularly generated, and the R-R distance was prolonged (cardiac rate, 20/min) at 12 min. The opercular rate declined to 64/min from the initial count of 90/min. (E) The profile at 21min was similar to (D), but the R-R distance was somewhat longer. J. Nutr. Sci. Vitaminol.
BLOOD GAS OF CO2 ANESTHETIZED CARP 39 oxygen saturation curve is similar to the PaO2 curve, reflecting the fact that oxygen saturation depends strongly on PaO2. Thus, the question whether PaO2 (or oxygen saturation) increases or decreases seems to depend on the levels of PaCO2 during anesthesia. Moderate levels of PaCO2 are essential to the maintenance of a safe level of sedation, and moderate levels of PaO2 are simultaneously essential to the survival of fishes. Figure 3 shows significant changes in ECG of carp under carbon dioxide anesthesia. When carp lay on their sides, a significant change in the QRS-complex appeared, from the R wave type to the RS wave type. A deficiency of RS wave sometimes appeared. Coupled with these observations, a gradual decline of the cardiac rate eventually caused arrhythmia and bradycardia (20beats/min in (D) in Fig. 3). This fall in the pulse rate was also accompanied by a fall in the opercular rate. This observation may support the idea of the existence of a connection between the cardiac and respiratory (opercular) centers in the brain as postulated by Randall (8). Subsequent ECG profiles were changed only slightly while the carp were lying on their sides. In conclusion, PaCO2 may have an influence on PaO2 or vice versa. The PaO2 - PaCO2 balance appears to be important in maintaining the long periods of sedation necessary for live fish to be transported safely. We understand that an ECG profile continues without significant change when carp become acclimated to an appro priate PaO2-PaCO2 balance. REFERENCES 1) Mitsuda, H., Kawai, F., Kuga, M., and Yamamoto, A. (1973): Mechanism of carbon dioxide gas absorption by grains and its application to skin-packaging. J. Nutr. Sci. Vitaminol., 19, 71-83. 2) Mitsuda, H., and Yasumatsu, K. (1955): Crystallization of animal catalase and studies on its optimum temperature. Bull. Agric. Chem. Soc. Jpn., 19, 200-207. 3) Mitsuda, H., Kawai, F., and Yamamoto, A. (1972): Underwater and underground storage of cereal grains. Novel storage system and packaging technique maintain quality during prolonged storage. Food Technol., 26, 50-56. 4) Mitsuda, H., Kawai, F., Yasumoto, K., and Hirotani, K. (1958): Effects of carbon dioxide on catalase. Bull. Inst. Chem. Res. Kyoto Univ., 36, 145-155. 5) Mitsuda, H., Nakajima, K., Mizuno, H., Kawai, F., and Yamamoto, A. (1980): Effects of carbon dioxide on carp. J. Nutr. Sci. Vitaminol., 26, 99-102. 6) McFarland, W. W. (1959): A study of the effects of anesthetics on the behavior and physiology of fishes. Publ, Inst. Marine Sci. (Texas), 6, 23-55. 7) Krogh, A., and Leitch, I. (1919): The respiration function of the blood of fishes. J. Physiol. Lond., 52, 288-300. 8) Randall, D. J. (1962): Effects of an anesthetic on the heart rate and respiration of Teleost fish. Nature, 195, 506-508. Vol. 28, No. 1, 1982