Camp. Biochem. Physiol., 1975, Vol. 51A, pp. 43 to 52. Pergamon Press. Printed in Great Britain
CIRCULATORY RESPONSES OF RAIVA CATESBEIANA TO TEMPERATURE, SEASON AND PREVIOUS THERMAL HISTORY WESLEY W. WEATHERS Department
of Environmental
Physiology, Rutgers-The State University, New Brunswick, New Jersey 08903, U.S.A. (Received 28 January 1974)
Abstract-l. Heart rate, femoral arterial pressure, plasma volume, blood volume and hematocrit ratio were determined on unrestrained, unanesthetized winter (January-March) and summer (July-August) bullfrogs which had been acclimated to 5 or 20°C for 2-3 weeks. 2. Winter frogs exhibited an increased sensitivity of heart rate to temperature change following cold acclimation. Heart rates of warm and cold acclimated summer frogs were not significantly different at comparable body temperatures (T,,‘s). 3. Femoral arterial pressure increased continuously with Tb between 0 and 36°C in warm acclimated winter and summer frogs, but tended to remain constant (winter frogs) or increased more slowly (summer frogs) over the same range of Tn’s in cold acclimated animals. 4. Cold acclimation had no effect on arterial capacitance measured in vitro. Arterial capacitance decreased markedly (a manifestation of increased vessel rigidity) at pressures above 30-40 mm Hg. 5. While blood volume did not vary with season or acclimation temperature, plasma volume was significantly reduced in cold acclimated winter frogs. 6. The hematocrit ratio increased following acclimation to cold in winter but not in summer.
INTRODUCTION
MATERIALS AND METHODS
UNLIKE most birds and mammals which maintain relatively stable internal temperatures, temperate zone amphibians undergo seasonal as well as daily fluctuations in body temperature. Metabolic compensations for changes in temperature, season and previous thermal history are well documented in many species of amphibians (reviews by: Adolph, 1951; Bullock, 1955; Fromm & Johnson, 1955; Precht, 1958 ; Prosser & Brown, 1962; Brown, 1965 ; Bishop & Gordon, 1967). When measured at comparable temperatures the rate of oxygen consumption is greater in summer than winter, and higher in cold than warm acclimated amphibians. Since tissue oxygen demands must be met by the product of cardiac output and A-V oxygen difference, circulatory adjustments to season, temperature and previous thermal history are to be expected. However, in contrast with the substantial literature concerning the circulatory adjustments of birds and mammals to these three parameters (reviews by: Lyman & Dawe, 1960; Sjiistrand, 1962; Lyman, 1965; Thauer, 1967a, b; Sturkie, 1970) relatively few studies have dealt with ectothermic vertebrates. The present study examines the effect of temperature, season and previous thermal history on several circulatory parameters, including heart rate and arterial blood pressure, in the anuran amphibian Rnna catesbeiana.
The circulatory responses of large (300-7OOg) adult bullfrogs, Rana catesbeiana, acclimated to $20 and 30°C during the winter (January-March) were compared with summer frogs (July-August) acclimated to 5 and 20°C. The animals were obtained from a commercial supplier (Jacques Weil Co., Rayne, Louisiana) and were captured near Rayne, Louisiana, a few days prior to shipment to New Brunswick, New Jersey. The animals were initially held in a large tank containing tap water at 20-23°C for 24-48 hr to permit them to recover from transport before being weighed and divided into groups containing frogs of similar size. For acclimation to a given temperature groups of nine to twelve frogs were placed in large stainless steel or enamel aquaria containing several cm of tap water which was changed every 24-48 hr. The animals were maintained in the dark at the desired temperature (+ 2°C) for 2-3 weeks prior to study. Frogs acclimated to 20 and 30°C were periodically force fed ground beef to maintain their original body weight, while frogs maintained at 5°C were not fed. Following the acclimation period some animals were used for determination of the relation of heart rate and arterial pressure to body temperature, (T’), while others were employed for estimates of blood volume and arterial capacitance. Femoral arterial pressures were measured from chronically implanted cannulas with Statham pressure transducers and recorded with a Grass Model 7A polygraph. From the pressure trace were taken heart rate, systolic, diastolic and pulse pressures. Measurements were made on unanesthetized, unrestrained frogs which were at thermal equilibrium and resting quietly in 43
WESLEYW. WEATHERS
44
the dark in individual 5-l. aquaria which contained several cm of tap water. Stable Tb’s between 0 and 36°C were obtained by placing the aquaria in a Honeywell Hotpack environmental chamber, and core Tb was measured to the nearest O.l”C with an intestinal thermocouple. A total of three to six measurements was made on each animal such that the data were evenly distributed over the temperature range 0-36°C. Plasma volume was determined on restrained unanesthetized animals by the dye dilution technique with the vital dye T-1824. For determination of plasma volume, the femoral artery was cannulated with a dry heparinized cannula and a blood sample immediately collected. This sample, whose volume was equal to the volume of dye to be injected, was used for: (a) determining the hematocrit ratio, (b) a plasma blank and (c) in some cases, erythrocyte count. The hematocrit ratio was determined as per cent packed cell volume by centrifuging duplicate samples of blood in microhematocrit tubes for 15 min in an
carefully checked for leaks and individual determinations yielded pressures which varied by less than 5 per cent. Determinations of the static pressure-volume relationship were usually conducted at a temperature of 20°C. However, in a few cases the influence of temperature on capacitance was determined by conducting measurements at temperatures between 0 and 40°C. The ratio of males to females was approximately equal in all groups, with males outnumbering females 2 to 1. There were no apparent differences between males and females with respect to any of the measured parameters. The slopes of regression lines fitted to semilogarithmic plots of heart rate vs. T,, were compared by a r-test (Steel & Torrie, 1960), while the regression lines for 5 and 20°C summer frogs were analyzed by a r-test which compares the intercepts of two lines having the same slope (Anderson & Bancroft, 1952). All other comparisons were by analysis of variance for two groups (Sokal & Rohlf, 1969).
Adams centrifuge. The erythrocyte number was determined as the average of three counts with a hemocytometer cell. For determination of plasma volume the dye,
RESULTS
in a concentration of 10 mg/kg body weight, was introduced into the circulation via an ipsolateral femoral venous cannula. Any dye which remained in the venous cannula was immediately flushed into the circulation with approximately 1 cm3 of previously withdrawn blood. After a IO-min mixing period, a sample of blood was withdrawn from the arterial cannula, the plasma fraction separated by centrifugation and the plasma concentration of T-1824 determined calorimetrically at a wavelength of 620 rnp with a Beckman Model B spectrophotometer. Measurements of blood volume were conducted at T,,‘s between 20 and 23°C. Following the determination of blood volume the animal was sacrificed by pithing, the right aortic arch removed for determination of arterial capacitance and total body water content was determined by drying the carcass to constant weight at 80°C. Arterial capacitance of winter frogs was determined on segments of the right aortic arch from which the connective tissue sheath was removed. The vessel was mounted at in situ length in a bath of 0.9% saline and one end of the vessel was connected by a polyethylene cannula to a Statham pressure transducer and the other end to a lOO-~1 syringe. The volume of the vessel was determined to the nearest 1 ~1 by observing the volume of air required to displace the fluid contained in the lumen. Known volumes of 0.9% saline were then injected into the vessel and resulting pressure recorded. The preparation was
The responses of winter bullfrogs acclimated to 20 or 30°C were not significantly different. Hence, for ease of presentation, data for winter frogs acclimated to 30°C are included in Table 1, but not in the figures. Throughout the text the responses of only four groups, frogs acclimated to 5 or 20°C during the winter and summer (henceforth referred to as 5 or 20°C winter frogs and 5 or 20°C summer frogs) will be compared. However, it should be understood that statements concerning 20°C winter frogs apply to 30°C winter frogs as well. Heart rate and arterial pressure The relation of resting heart rate to Tb was exponential in all groups. Lines fitted to semilogarithmic plots of heart rate vs. T,,, by the method of least squares, have coefficients of correlation which are highly significant (P
CIRCULATORY RESPONSES OF RAIVA CATESBEIANA TO TEMPERATURE, SEASON AND PREVIOUS THERMAL HISTORY WESLEY W. WEATHERS Department
of Environmental
Physiology, Rutgers-The State University, New Brunswick, New Jersey 08903, U.S.A. (Received 28 January 1974)
Abstract-l. Heart rate, femoral arterial pressure, plasma volume, blood volume and hematocrit ratio were determined on unrestrained, unanesthetized winter (January-March) and summer (July-August) bullfrogs which had been acclimated to 5 or 20°C for 2-3 weeks. 2. Winter frogs exhibited an increased sensitivity of heart rate to temperature change following cold acclimation. Heart rates of warm and cold acclimated summer frogs were not significantly different at comparable body temperatures (T,,‘s). 3. Femoral arterial pressure increased continuously with Tb between 0 and 36°C in warm acclimated winter and summer frogs, but tended to remain constant (winter frogs) or increased more slowly (summer frogs) over the same range of Tn’s in cold acclimated animals. 4. Cold acclimation had no effect on arterial capacitance measured in vitro. Arterial capacitance decreased markedly (a manifestation of increased vessel rigidity) at pressures above 30-40 mm Hg. 5. While blood volume did not vary with season or acclimation temperature, plasma volume was significantly reduced in cold acclimated winter frogs. 6. The hematocrit ratio increased following acclimation to cold in winter but not in summer.
INTRODUCTION
MATERIALS AND METHODS
UNLIKE most birds and mammals which maintain relatively stable internal temperatures, temperate zone amphibians undergo seasonal as well as daily fluctuations in body temperature. Metabolic compensations for changes in temperature, season and previous thermal history are well documented in many species of amphibians (reviews by: Adolph, 1951; Bullock, 1955; Fromm & Johnson, 1955; Precht, 1958 ; Prosser & Brown, 1962; Brown, 1965 ; Bishop & Gordon, 1967). When measured at comparable temperatures the rate of oxygen consumption is greater in summer than winter, and higher in cold than warm acclimated amphibians. Since tissue oxygen demands must be met by the product of cardiac output and A-V oxygen difference, circulatory adjustments to season, temperature and previous thermal history are to be expected. However, in contrast with the substantial literature concerning the circulatory adjustments of birds and mammals to these three parameters (reviews by: Lyman & Dawe, 1960; Sjiistrand, 1962; Lyman, 1965; Thauer, 1967a, b; Sturkie, 1970) relatively few studies have dealt with ectothermic vertebrates. The present study examines the effect of temperature, season and previous thermal history on several circulatory parameters, including heart rate and arterial blood pressure, in the anuran amphibian Rnna catesbeiana.
The circulatory responses of large (300-7OOg) adult bullfrogs, Rana catesbeiana, acclimated to $20 and 30°C during the winter (January-March) were compared with summer frogs (July-August) acclimated to 5 and 20°C. The animals were obtained from a commercial supplier (Jacques Weil Co., Rayne, Louisiana) and were captured near Rayne, Louisiana, a few days prior to shipment to New Brunswick, New Jersey. The animals were initially held in a large tank containing tap water at 20-23°C for 24-48 hr to permit them to recover from transport before being weighed and divided into groups containing frogs of similar size. For acclimation to a given temperature groups of nine to twelve frogs were placed in large stainless steel or enamel aquaria containing several cm of tap water which was changed every 24-48 hr. The animals were maintained in the dark at the desired temperature (+ 2°C) for 2-3 weeks prior to study. Frogs acclimated to 20 and 30°C were periodically force fed ground beef to maintain their original body weight, while frogs maintained at 5°C were not fed. Following the acclimation period some animals were used for determination of the relation of heart rate and arterial pressure to body temperature, (T’), while others were employed for estimates of blood volume and arterial capacitance. Femoral arterial pressures were measured from chronically implanted cannulas with Statham pressure transducers and recorded with a Grass Model 7A polygraph. From the pressure trace were taken heart rate, systolic, diastolic and pulse pressures. Measurements were made on unanesthetized, unrestrained frogs which were at thermal equilibrium and resting quietly in 43
WESLEYW. WEATHERS
44
the dark in individual 5-l. aquaria which contained several cm of tap water. Stable Tb’s between 0 and 36°C were obtained by placing the aquaria in a Honeywell Hotpack environmental chamber, and core Tb was measured to the nearest O.l”C with an intestinal thermocouple. A total of three to six measurements was made on each animal such that the data were evenly distributed over the temperature range 0-36°C. Plasma volume was determined on restrained unanesthetized animals by the dye dilution technique with the vital dye T-1824. For determination of plasma volume, the femoral artery was cannulated with a dry heparinized cannula and a blood sample immediately collected. This sample, whose volume was equal to the volume of dye to be injected, was used for: (a) determining the hematocrit ratio, (b) a plasma blank and (c) in some cases, erythrocyte count. The hematocrit ratio was determined as per cent packed cell volume by centrifuging duplicate samples of blood in microhematocrit tubes for 15 min in an
carefully checked for leaks and individual determinations yielded pressures which varied by less than 5 per cent. Determinations of the static pressure-volume relationship were usually conducted at a temperature of 20°C. However, in a few cases the influence of temperature on capacitance was determined by conducting measurements at temperatures between 0 and 40°C. The ratio of males to females was approximately equal in all groups, with males outnumbering females 2 to 1. There were no apparent differences between males and females with respect to any of the measured parameters. The slopes of regression lines fitted to semilogarithmic plots of heart rate vs. T,, were compared by a r-test (Steel & Torrie, 1960), while the regression lines for 5 and 20°C summer frogs were analyzed by a r-test which compares the intercepts of two lines having the same slope (Anderson & Bancroft, 1952). All other comparisons were by analysis of variance for two groups (Sokal & Rohlf, 1969).
Adams centrifuge. The erythrocyte number was determined as the average of three counts with a hemocytometer cell. For determination of plasma volume the dye,
RESULTS
in a concentration of 10 mg/kg body weight, was introduced into the circulation via an ipsolateral femoral venous cannula. Any dye which remained in the venous cannula was immediately flushed into the circulation with approximately 1 cm3 of previously withdrawn blood. After a IO-min mixing period, a sample of blood was withdrawn from the arterial cannula, the plasma fraction separated by centrifugation and the plasma concentration of T-1824 determined calorimetrically at a wavelength of 620 rnp with a Beckman Model B spectrophotometer. Measurements of blood volume were conducted at T,,‘s between 20 and 23°C. Following the determination of blood volume the animal was sacrificed by pithing, the right aortic arch removed for determination of arterial capacitance and total body water content was determined by drying the carcass to constant weight at 80°C. Arterial capacitance of winter frogs was determined on segments of the right aortic arch from which the connective tissue sheath was removed. The vessel was mounted at in situ length in a bath of 0.9% saline and one end of the vessel was connected by a polyethylene cannula to a Statham pressure transducer and the other end to a lOO-~1 syringe. The volume of the vessel was determined to the nearest 1 ~1 by observing the volume of air required to displace the fluid contained in the lumen. Known volumes of 0.9% saline were then injected into the vessel and resulting pressure recorded. The preparation was
The responses of winter bullfrogs acclimated to 20 or 30°C were not significantly different. Hence, for ease of presentation, data for winter frogs acclimated to 30°C are included in Table 1, but not in the figures. Throughout the text the responses of only four groups, frogs acclimated to 5 or 20°C during the winter and summer (henceforth referred to as 5 or 20°C winter frogs and 5 or 20°C summer frogs) will be compared. However, it should be understood that statements concerning 20°C winter frogs apply to 30°C winter frogs as well. Heart rate and arterial pressure The relation of resting heart rate to Tb was exponential in all groups. Lines fitted to semilogarithmic plots of heart rate vs. T,,, by the method of least squares, have coefficients of correlation which are highly significant (P
