Ventilation in An Aquatic and A Terrestrial Chelonian Reptile

The tortoise Testudo and the turtle Pelomedusa exhibit two different modes of breathing. Periods of breath-holding are typical of the normal breathing pattern in both species, but such periods are interrupted by episodes of continuous breathing in Pelomedusa, whereas single breaths and short periods of breath-holding alternate in Testudo. The periodic breathing pattern of Pelomedusa is typical of aquatic reptiles, and persists both in Pelomedusa and in other species when they leave the water (Burggren, 1975).

Periodic ventilation suggests that and in both the alveolar gas and blood will change in a cyclic manner (Lenfant et al. 1970). The fluctuations in blood gas tensions in chelonians are also complicated by intracardiac blood shunting. A net left-to-right shunt normally occurs during the period of ventilation, and a net right- to-left shunt of blood away from the lungs increases during apnoea (Millen et al. 1964; White & Ross, 1965, 1966; Johansen, Lenfant & Hanson, 1970; Shelton & Burggren, 1976).

Nearly every study on the control of ventilation in reptiles during hypoxia or hypercapnia has been deficient in measurements of blood and alveolar and (Wood & Lenfant, 1976). Exceptions are the studies on control of ventilation in the freshwater turtle, Pseudemys scripta elegans, in which blood gas tensions were measured (Frankel et al. 1969; Jackson, Palmer & Meadows, 1974). However, measurements in these studies did not provide information on the time needed for the arterial or to attain stable values during hypoxia or hypercapnia.

The present paper deals with the relationship of ventilation to lung (end-tidal) in two chelonian species, the terrestrial tortoise Testudo pardalis, and an aquatic species, the side-necked turtle Pelomedusa subrufa. The time course for development of the ventilatory response during hypercapnia was measured in both species. This was compared with the time course of increase of end-tidal in Testudo, while rate of increase of blood was obtained for Pelomedusa. The effect of changing inspired on ventilation was measured in both species and reference values on the normal patterns of breathing were obtained. Air convection requirements were also studied. The air convection requirement of an animal is the volume of air which must ventilate the gas exchange surfaces to extract a unit amount of oxygen (Dejours, 1972).

Four leopard tortoises, Testudo pardalis, and five side-necked turtles, Pelomedusa subrufa, were airfreighted from Kenya to Aarhus, Denmark. The tortoises weighed from 2 to 3·5 kg (mean 2·9 kg), and the turtles from 0·3 to 0·8 kg (mean 0·57 kg). The tortoises were fed a varied diet of fresh vegetables, whereas the turtles were offered liver and fish meat. The animals readily accepted the food and were in excellent condition. Animals were not fed for 2 days prior to experiments. All measurements were performed at 25 °C, which is within the range of the preferred temperatures for both species, and the animals were kept at that temperature between experiments.

A skin-tight mask of flexible polymer was constructed for each animal (see Glass, Wood & Johansen, 1977, for details). The mask was glued to the head of the animal using a rapidly setting epoxy glue. Mask and glue covered the front of the head and the mouth, but the eyes were unobstructed. A light-weight Fleisch head (Fleisch, 1925) connected to a Godart pneumotachograph was inserted into an opening in the mask over the nostrils. Calibration was performed as advised by Hobbes (1967). The mask with the inserted Fleisch head did not prevent the animal from retracting its head.

During experiments the animals were placed in a plexiglass chamber which permitted observation of the animal. The outlet tube served additionally for passing the pressure and gas sampling tubes from the face mask to pneumotach and gas analysers. The dimensions of the chamber restricted movements of the animal, but did not interfer with its natural posture.

When ventilation of a Pelomedusa was measured the experimental chamber was partly filled with water, but only to a level at which the animal was able to submerge its head. Unlike Testudo, which readily tolerated face masks, it took several days to condition a Pelomedusa to wear a mask. Ventilation was measured in two different ways for this reason. The alternative was the buoyancy technique described by Jackson (1971). With this technique, the turtle was suspended by strings from a Grass strain gauge transducer. The body was lowered into water, but the turtle could raise its head to the surface for breathing. Lung inflation and deflation caused changes in the buoyancy of the animal which were monitored by the strain gauge transducer and a recorder (Brush 260). The accuracy of this method of measuring tidal volume is very high and is set by the sensitivity of the strain gauge transducer. Its successful application, however, depends on minimum restraint of the turtles since the mechanics of ventilation require relatively free leg movements. Fortunately the Pelomedusa were usually quiescent during measurements and only needed to be loosely restrained. The two methods of measuring ventilation were in excellent agreement, differing by less than 5%.

CO₂ contents of gases were measured by a Beckman LB₂ CO₂ analyser which was calibrated with a Wosthoff gas mixing pump fed with pure gases. The sampling rate of the LB2 was reduced to about 60 ml. min⁻¹. This reduction increased the lag and rise time of the LB2, but it was possible in every case to obtain an ‘alveolar plateau’ during expiration. The sample cell of the LB2 was frequently cleaned to assure linear performance. Expired gas was sampled on a breath-to-breath basis through a catheter terminating in a hypodermic needle that penetrated the mask close to the nostrils. Sampling from this point minimized dead space.

of hypoxic gases was measured by means of a Radiometer O₂ electrode mounted in a cuvette. The was continuously displayed on a Radiometer O₂ module. of a single breath could not be measured by the O₂electrode, but was measured by a Medspect 2 respiratory mass-spectrometer, which was used in ten experiments to obtain end-tidal and ⁠. Information on both gases is needed for the calculation of the air convection requirements.

Hypercapnic or hypoxic gas mixtures were prepared by feeding gases from cylinders through Fisher and Porter flowmeters into a water-filled wash bottle where mixing took place. The experimental chamber was continuously flushed with the mixture at a rate of 2–3 l.min⁻¹. The partial pressures of CO₂ in the test gases were 14·7, 29·4 and 44·2 mmHg corresponding to 2, 4 and 6% of the dry gas content at 25 °C and 1 atmosphere. Partial pressures of the hypoxic or hyperoxic gases were 37, 74, 155 and 736 mmHg at the same temperature and barometric pressure.

The methods of intravascular cannulation were described in detail by Shelton & Burggren (1976) and Burggren, Glass & Johansen (1977). The common pulmonary artery and the left subclavian artery were non-occlusively cannulated in an upstream direction. Sampling of blood was not started until 24 h after surgery. The blood samples were analysed with Radiometer equipment (BMS 3, PHM 71). The total accumulated volume from repeated sampling ranged between 3·3 and 5·1 ml/kg.

An animal was left undisturbed in the experimental chamber for 4–7 h (mean 5 h) after glueing of mask and Fleisch head to the animal. Test gases were introduced when a stable minimum ventilation had been observed. Composition of the inspired gas could be altered within half a minute. Inhalation of the test gas usually lasted about 1 h. The animal was allowed to recover in atmospheric air before another test gas was introduced.

The peak of the expired gas is the end-tidal ⁠, which was used as an approximation to the mean alveolar ⁠. All gas rates are calculated as ml. min⁻¹. kg⁻¹, but conventionally the inspired minute volume and the alveolar minute volume are at body temperature and pressure, saturated with water vapour (BTPS), whereas volumes of O₂ and CO₂ are calculated for standard con-ditions (STPD = 0 °C, 760 mmHg, dry). The ratio was calculated by means of the equation where R is the gas constant equal to 2·785 ml.mmHg. °K⁻¹.ml⁻¹ STPD. Air convection requirements could be calculated from the respiratory quotient of the expired air and the ratio ⁠. Expired volumes were less than inspired volumes in Pelomedusa due to CO₂ elimination to the water. The ventilations discussed in the following pages are based on inspired volumes ⁠, since expired volumes are not directly comparable in Pelomedusa and Testudo due to a marked difference in their pulmonary gas exchange ratios.

Fig. 1 shows recorded tracings of representative breathing patterns of Testudo and Pelomedusa. Periods of breath-holding and single breaths alternated in Testudo, whereas breathing in Pelomedusa vias characterized by episodes of several consecutive breaths separated by periods of breath-holding. In both species breath-holding was preceded by an inspiration and resumption of breathing was initiated by an expira-tion. A ventilatory period in Pelomedusa usually consisted of four to seven breaths, but breath-holding was of variable duration (mean of five animals being 4·3 min.) (Fig. 8). This is about six times the mean interval between breaths for four Testudo (0·7 min). Table 1 compares tidal volume, respiratory frequency and ventilation in both species. It is based on periods that include about 50 breaths and thus several episodes of ventilatory activity for each animal. Therefore, the frequencies express long-term values and do not reflect the temporal spacing between individual breaths.

The table also shows end-tidal ⁠, respiratory quotients, and air convection require-ments for both species and in addition blood gas tensions in Pelomedusa. It is evident from the table that overall respiratory frequencies of the two species were not signifi-cantly different in spite of the differences in breathing pattern. Frequency was only slightly above 1 breath/min in both species. The mean weight-specific tidal volume in Pelomedusa (15·5 ml/kg) was larger than in Testudo (10·7 ml/kg), but the variation between the individual animals was large in both species. The mean end-tidal in Pelomedusa was two-thirds of that in Testudo (Fig.2). The and in the tortoise were very stable when breathing was regular. In contrast the periodic mode of breathing in Pelomedusa caused a progressive decrease of and increase of ⁠, with each breath during ventilation (Fig. 2). The mean respiratory quotient calculated on the basis of expired air composition was 0·76 in Testudo but only 0·52 in Pelomedusa. The latter value reflects aquatic CO₂ elimination which is of variable importance in aquatic turtles (Belkin, 1963). Air convection requirements were not significantly different in Pelomedusa (27·3 ml BTPS/ml STPD) and Testudo (25·4 ml BTPS/ml STPD) in spite of the differences in patterns of breathing. The importance of the blood gas levels in normal control of breathing was difficult to assess both because of the small number of samples and their arbitrary timing in relation to the breathing cycle. Subclavian arterial was approximately 50 mmHg higher than pulmonary arterial in all Pelomedusa, a situation similar to that in other chelonian reptiles with a functionally undivided ventricular pump (Burggren, 1976). averaged 18·3 mmHg, compared with 23·3 mmHg in the pulmonary artery (Table 1).

Ventilation was increased by hypoxia and depressed by hyperoxia in both species (Fig. 3). Lowering of the inspired from normal (about 155 mmHg) to 37 mmHg increased ventilation by a factor of 2·9 in Pelomedusa and 1·9 in Testudo. The responses were produced by variable changes in both tidal volume and frequency, but a con-sistent change of these variables was only observed in two cases. One Pelomedusa consistently increased frequency when was decreased from 736 to 37 mmHg, whereas a tortoise consistently increased tidal volume. The end-tidal in the tortoises decreased to 25−28 mmHg, when was 37 mmHg. The same caused the in Pelomedusa to drop from the normal average value of 70 mmHg to 31 mmHg (range: 25−35 mmHg), while the average of the pulmonary artery dropped from 33·7 to 22·3 mmHg (range: 16-29 mmHg).

The time course of the ventilation increase during hypercapnia in Testudo was correlated with the rate of increase of end-tidal ⁠, but the ventilation increase in Pelomedusa was not in simple proportion to blood ⁠. Fig. 4 shows the time course of the increase of end-tidal and ventilation during hypercapnia for three speci-mens of Testudo. The increases are shown as percentages of the maximum increases. Mean was 33 mmHg but still increased slightly, even after nearly an hour of CO₂ breathing. The increase of ventilation reached a maximum (mean 206 ml. kg⁻¹. min⁻¹) after about 40 min. The time course of blood and the time-response characteristics of ventilation during hypercapnia are shown in Fig. 5 for three specimens of Pelomedusa. Changes in ventilation are averaged for 10 min periods to eliminate the irregularities caused by periodic ventilation, which persisted even during hyperventilation. in the pulmonary artery and the left subclavian artery increased steadily during all 30 min of the experiment, but on the average 85% of the ventilation increase occurred during the first 10 min of the experiment, with a maximum at 10–20 min after the start of CO₂ breathing.

Fig. 6 shows the difference between end-tidal and inspired ⁠, plotted against inspired for four specimens of Testudo. The differences are those that occur after 40 min of CO₂ breathing, and are much reduced at high inspired .

The relationships between end-tidal ⁠, tidal volume, respiratory frequency, and ventilation are shown in Figs. 7–9. The low values are those of the normal stable breathing pattern. The increases of ventilation are induced by inspiration of 2,4 and 6 % CO₂ (dry gas content). The figures are based on the conditions during maximum response to the test gases. The responses of the individual animals are presented for Testudo. Tidal volume and frequency increased in proportion to end-tidal up to about 35 mmHg, but the effect of increasing to about 45 mmHg was less than would be expected from an extrapolation of the response to an increase of up to 35 mmHg. The frequency increase was less consistent than the increase of tidal volume. The increase of ventilation per unit increase of ( ml BTPS. kg⁻¹.min⁻¹,mmHg⁻¹) ranged widely, from 4·7 to 35·1.

The responses of Pelomedusa are presented as grouped data and include measure-mente of ventilation in five specimens ; expired ⁠, was measured in two of these. The ⁠, is an average including all expirations. Both tidal volume, frequency, and the resulting ventilation appear to increase approximately in proportion to the end-tidal ⁠, up to a value of 30 mmHg. These relationships would not result if the mean ⁠, of all expirations was replaced by the mean value for the expirations that initiate the ventilatory periods. This mean value is 17 mmHg during normal stable breathing, which would shift the pointe for the normal breathing pattern to the right in Figs. 7–9. The other pointe would remain unchanged, because hypercapnia practically abolished the variation in end-tidal ⁠. It is worth noting that when ⁠, was increased to 44 mmHg the end-tidal of Pelomedusa was less than inspired ⁠. This reflects CO₂ elimination to the water.

In Pelomedusa, inspiration of 2 % CO₂ reduces the duration of the breath-holding periods to one-third of the initial mean value. Notably a much shortened breath-holding period persists at the 6% CO₂ level. By comparing Figs. 8(a) and (b), it is evident that the shortening of the breath-holding periods alone can not account for the frequency response to hypercapnia. The breathing frequency and the duration of the ventilatory periods are also modified.

A contrast exists between the periodic breathing pattern of Pelomedusa and the alternation between single breaths and short periods of breath holding in Testudo, but these differences are not matched by any significant differences in the long term respiratory frequency. Weight-specific tidal volume in Pelomedusa exceeds that of Testudo. This leads to a higher weight-specific ventilation in agreement with the relative weights of the two species. The respiratory frequency and tidal volume of Pelomedusa are very similar to those reported for Pseudemys (Jackson, 1971). The high weight-specific tidal volume of diving turtles relative to that of Testudo will reduce the number of breaths needed to change gas composition in the lungs after a diving period. In snakes, the weight-specific ventilation of an aquatic species is less than for terrestrial species. The aquatic species practising long breath holds (up to 1 h) compensate for this by possessing much smaller air convection requirements (Glass & Johansen, 1976). Similar tendencies in chelonians are not obvious in the| present data. Air convection requirements of Pelomedusa and Testudo at 25 °C at not significantly different and close to the requirements of Pseudemys (Jackson, 1971). The lower end-tidal in Pelomedusa than in Testudo reflects aquatic CO₂ elimina-tion. Cutaneous respiration lowers blood and conversely, as stated by Dejours (1975): ‘the more an animal is a pulmonary breather, the higher is its blood CO₂ pressure’. Aquatic CO₂ elimination also explains the relatively small fluctuations in lung and blood gas in spite of the occurrence of diving periods.

The hyperbolic relationship between inspired and ventilation in both Pelo-medusa and Testudo has earlier been reported for Pseudemys (Frankel et al. 1969; Jackson, 1973). In contrast Altland & Parker (1955) found that hypoxia influenced the pattern of breathing, but not the actual ventilation volume in Terrapene Carolina carotina. The duration of the diving period has been shown to depend on in several species of turtles, Terrapene (Altland & Parker, 1955), Chelydra serpentine (Boyer, 1963) and Chelys fimbriata (Lenfant et al. 1970). The increase of ventilation in Testudo and Pelomedusa was not correlated with any consistent pattern of changes in frequency or tidal volume, as was similarly reported for Pseudemys (Jackson, 1973).

The fact that blood in Pelomedusa had not reached a stable value in relation to the of the inspired hypercapnic gas even after 30 min (Fig. 4) supports the statement by Wood & Lenfant (1976) that data on control of ventilation in reptiles should be based on measurement of gas tensions in the blood and the lungs, and not on inspired gas tensions alone. There is no obvious explanation for the low rate of increase of blood in Pelomedusa. Intracardiac right-to-left shunting of blood away from the lungs could be an explanation, but Burggren et al. (1977) actually found hypercapnia to stimulate lung perfusion. The CO₂ diffusing capacity of the chelonian lung is low (Crawford et al. 1976) and thus could be partly responsible for the low rate of equilibration. A complication in the relationship between blood and ventilation is the fact that ventilation reached a maximum or even decreased, while blood continued to increase. Ventilation did not reach an absolute maxi-mum limit at 4% CO₂, since inspiration of 6% CO₂ could produce an additional increase of ventilation.

The results of the experiments on the relationship of ventilation to end-tidal are more clear. Ventilation and end-tidal increased at comparable rates in Testudo, except that ventilation reached a maximum level while end-tidal still increased, though very slowly. The fact that an increase of did not cease completely even after nearly 1 h of CO₂ breathing indicates that the ventilation in-crease is inadequate for eliminating the CO₂ produced by the animal in spite of an impressive increase of tidal volume. If lung volume of Testudo pardalis is comparable to a lung volume in Testudo graeca of 171 ml/kg (Crawford et al. 1976), then the ratio of tidal volume to total lung volume increases from 6% to slightly less than 30% during hypercapnia.

A simple relationship of ventilation to end-tidal was evident in Pelomedusa and Testudo, although complicated by some depression of ventilation at high CO₂. Ventilation increased in proportion to blood in Pseudemys (Jackson et al. 1974). The correlation between end-tidal and ventilation takes on interest in light of the recent evidence for the existence of intrapulmonary CO₂-sensitive receptors in the Tegu lizard (Gatz, Fedde, & Crawford 1975; Fedde, Kuhlmann & Scheid, 1977; Scheid, Kuhlmann & Fedde, 1977), and turtle (Milsom & Jones, 1976). The receptors are both mechano- and CO₂-sensitive in the turtle, but not in the Tegu lizard in which the discharge rate of the receptors increases when in the lung gas is reduced. Additionally, a sudden reduction of lung induces a period of breath-holding in the Tegu lizard (Gatz et al. 1975). This response suggests that the receptors are involved in a positive ventilatory response to an increase of lung ⁠, which may explain the fairly simple relationships between end-tidal and ventilation seen in Testudo and Pelomedusa. However, the possible role of other peripheral receptor systems in reptiles has not been adequately investigated (Wood & Lenfant, 1976). Although there is still much to be learned about ventilation and the periodic breathing pattern in turtles, it is evident from the present data that the stimuli affecting ventila-tion in terrestrial and aquatic species are not fundamentally different. It is also evident that both and may influence normal breathing in both Testudo and Pelo-medusa. The influence of on normoxic breathing is revealed by the fact that hyperoxia caused a depression of ventilation. The relative importance of and in the control of normal breathing is difficult to analyse. Wood & Lenfant (1976) discuss the evidence that hypoxia is important in the control of breathing in chelonians. Although the present data show a very sensitive ventilatory response to hypercapnia in both the aquatic and the terrestrial species, it should be noted that fluctuations of blood and lung gas in Pelomedusa are larger than fluctuations of which tend to be decreased by aquatic CO₂ elimination during normal breathing.

The authors acknowledge the generous supply of animals from Professor G. M. O. Maloiy, Nairobi, Kenya.