The accessory role of the diaphragmaticus muscle in lung ventilation in the estuarine crocodile Crocodylus porosus

Crocodilians generate subatmospheric pulmonary pressures to inflate their lungs. Unlike mammals, in which the diaphragm plays a central role, crocodilians lack a muscular structure homologous or analogous to the mammalian diaphragm and a combination of three other muscular mechanisms power ventilation; namely, the intercostal, abdominal and diaphragmaticus muscles.

Intercostal muscles are active during both inspiration and expiration (Gans and Clark, 1976). Inspiration is driven by cranial rotation of tripartite ribs, which increases thoracic volume whereas caudal and medial rotation of the ribs decreases thoracic volume during expiration (Claessens, 2009). The abdominal muscles act to alter abdominal volume either by displacing the liver cranially during expiration or by providing room for the caudal displacement of the liver during inspiration. The rectus abdominis and transversus abdominis muscles are active during inspiration and expiration (Gans and Clark, 1976; Naifeh et al., 1970), particularly during exercise (Farmer and Carrier, 2000a). The rectus abdominis muscle (and possibly the transversus abdominis) also rotate the pubic bones in the craniodorsal direction and contribute to decreasing abdominal volume during expiration (Farmer and Carrier, 2000a). The ischiopubis and ischiotruncus muscles act to increase abdominal volume during inspiration by rotating the pubic bones ventrally (Farmer and Carrier, 2000a).

The diaphragmaticus muscle of crocodilians is not homologous to the mammalian diaphragm (Gans, 1971; Klein and Owerkowicz, 2006) and its main function may have been non-respiratory (Uriona and Farmer, 2008) as crocodilian ancestors became secondarily adapted to life in water (Seymour et al., 2004). The diaphragmaticus has been well described in caiman and alligator (Boelaert, 1942; Claessens, 2009; Farmer and Carrier, 2000a; Gans and Clark, 1976; Naifeh et al., 1970; Uriona and Farmer, 2006). In alligators, the two-paired strap-like muscles originate on the ischia and on the last gastralia and insert onto a connective tissue sheath that surrounds the liver (Farmer and Carrier, 2000a). In caiman (Gans and Clark, 1976) and crocodiles (S.L.M., personal observation), the origin of the diaphragmaticus muscle differs slightly from that in alligators and encompasses the ischia and the pubis. Contraction of the diaphragmaticus muscle pulls the liver caudally, increasing thoracic volume and facilitating inspiration (Farmer and Carrier, 2000a; Gans, 1971; Gans and Clark, 1976; Naifeh et al., 1970). The caudocranial translation of the liver during the ventilatory cycle has been likened to a piston, and hence the term ‘hepatic piston pump’ has been coined to describe the mechanism powered by the diaphragmaticus muscle (Gans and Clark, 1976). The hepatic piston pumping has been shown to effectively decouple terrestrial locomotor mechanics from breathing mechanics in the American alligator (Farmer and Carrier, 2000b), and thus may provide a functional advantage during exercise compared with costal ventilation alone.

Previous studies have shown that lung ventilation in crocodilians can be effected by various combinations of muscular mechanisms. In submerged caiman, lung ventilation was achieved solely by use of the hepatic piston pump (Gans and Clark, 1976) with costal muscle activity being neither regular nor obligatory (Gans, 1971). In juvenile alligators on land, lung ventilation was achieved by a combination of both costal and hepatic piston mechanisms (Farmer and Carrier, 2000a). These studies suggest that the diaphragmaticus muscle plays a primary role in inspiration. This argument is further supported by recent videoradiographic measurements of lung volume in resting alligators (Claessens, 2009), where the diaphragmatic contribution to lung inflation has been determined to range from 36% to 61% of inspired tidal volume.

Evidence that the diaphragmaticus muscle is not absolutely necessary for effective lung ventilation at rest has been demonstrated in hatchling and juvenile alligators with a surgically transected diaphragmaticus (Hartzler et al., 2004; Uriona and Farmer, 2006). The loss of diaphragmatic function was found to result in significant reductions in maximal inspiratory flow rate; however, the effect on respiratory gas exchange was not quantified.

The goal of our study is to determine the inspiratory importance of the diaphragmatic muscle in juveniles of the estuarine crocodile (Crocodylus porosus). Extant crocodilians genera show differences in their habitat and activity preferences (Webb et al., 1993), thus the relative contribution of the diaphragmaticus muscle to lung ventilation may vary between groups. So far, however, only Alligator and Caiman have been studied from this perspective. In contrast to previous studies at a single temperature and at rest, we measured the contribution of the diaphragmaticus muscle to lung ventilation, and its effect on gas exchange, in crocodiles under altered respiratory demand associated with decreased body temperature, recovery from forced exercise and hypercapnia.

Five estuarine crocodiles (Crocodylus porosus Schneider 1801) of indeterminate sex were obtained from the Koorana Crocodile Farm, Rockhampton, Australia, and kept in aquaria with a thermal gradient (27–33°C), full spectrum lighting (14 h:10 h light:dark), free access to water and were fed a diet of whole rodents, fish and chicken pieces. Body mass ranged from 0.60 kg to 1.42 kg (mean ± s.e.m., 0.98±0.19 kg). Experiments were conducted under institutional animal ethics permit [LTU 04/37(L)].

Crocodiles were anaesthetised with halothane (Veterinary Companies of Australia, Artarmon, NSW, Australia), intubated and artificially ventilated (Model 661, Harvard Apparatus, Millis, MA, USA) with room air that had been passed through a vapourizer (Fluotec 3, Cyprane Limited, Keighley, Yorkshire, UK). The vapourizer was initially set at 4–5% for the induction of anaesthesia, and was then reduced to 1–2% for surgical maintenance. An incision was made in the skin and cervical muscles were carefully blunt-dissected to expose the underlying carotid artery. The carotid artery was cannulated with heparinised polyethylene tubing (i.d. 0.023 mm, o.d. 0.038 mm; Microtube Extrusions, North Rocks, NSW, Australia) and the tubing was looped once prior to exiting the wound where it was secured to the skin using two sutures. The incision site was closed with silk sutures.

Electromyography (EMG) electrodes (0.05 mm diameter copper wire) were inserted bilaterally (and perpendicular to muscle fibre orientation) into the diaphragmaticus muscle via a 3–4 cm midline abdominal incision. A copper ground electrode (with frayed ends) was also placed in the abdominal cavity. Leads from the electrodes were subcutaneously tunnelled to a dorsal exit just caudal to the hindlimb. All incisions were closed with interrupted sutures and treated with cyanoacrylate tissue adhesive (Vetbond, 3M, St Paul, MN, USA). The cannula and lead wires were coiled and taped to the back of the animal. Artificial ventilation with room air was continued until the crocodile regained consciousness and initiated spontaneous breathing. Intramuscular injections of the antibiotic Duplocillin (Intervet Australia, Bendigo East, VIC, Australia) and the analgesic Temgesic (Buprenophine, Reckitt Benckiser, West Ryde, NSW, Australia) were given at the conclusion of surgery. Duplocillin injections were repeated every second day after surgery. A minimum recovery period of 2 days was allowed before experiments commenced.

After the first set of experiments, crocodiles were anaesthetised for a second time as described above. The diaphragmaticus muscle was exposed via the previous incision site, and transected by surgically severing the muscle bellies from their origin on the pubis and the ischia. After, the incision was closed and animals recovered as described above. Complete transection of the diaphragmaticus muscle was confirmed for each animal by post-mortem examination at the end of the study.

Breathing patterns were analysed in terms of tidal volume (V_T), breathing frequency (f), minute ventilation (V̇_E=V_T×f), inspiratory and expiratory durations (T_I and T_E), the duration of the non-ventilatory period (T_NVP), rate of inspiratory airflow (VT_I/T_I), air convection requirements for O₂ (ACR O₂ =V̇_E/V̇_O2) and CO₂ (ACR CO₂=V̇_E/V̇_CO2) and respiratory exchange ratio (RER=V̇_CO2/V̇_O2). For each test condition, a mean of 40 consecutive breaths were analysed, and ventilatory volumes are reported at body temperature and barometric pressure, saturated (BTPS).

The arterial blood partial pressures of O₂ (Pa_O2) and CO₂ (Pa_CO2) and pH were measured with BMS 3 Mk 2 and PHM 73 (Radiometer, Brønshøj, Denmark), respectively, at the appropriate test temperature (20°C or 30°C) via small blood samples (250–300 μl) taken from the arterial cannula and stored anaerobically on ice. The electrodes were calibrated before and after each measurement. Pa_O2 and Pa_CO2 were measured every 30 s for 3 min and regressed back to time zero to account for drift and/or O₂ consumption by the electrode; pH was measured in incremental volumes of blood until the variation between successive measurements was less than 0.005 units. The arterial oxygen content (Ca_O2) of each blood sample was determined from a 10 μl subsample of blood using a galvanic cell (Oxygen Content Analyser, OxyCon, University of Tasmania, TAS, Australia). Lactate concentration was determined by an Accusport analyser (Boehringer Mannheim, Mannheim, Germany) and haemoglobin concentration by the HemoCue analyser (HemoCue AB, Ängelholm, Sweden). Note that neither analyser had been validated for use with reptile blood.

Electromyographic signals were amplified and recorded using a Powerlab data acquisition system (Model 8/30, AD Instruments) and analysed using Powerlab Chart Pro software (AD Instruments).

Crocodiles were fasted for 7 days prior to surgery (to ensure a post-absorptive state) and were held at 30°C for 2–3 days prior to experimentation (to ensure stable respiratory and metabolic parameters). At the time of the experiment the body temperature (T_B) of the crocodiles was monitored via a thermocouple inserted ∼5 cm into the cloaca (temperature pod, AD Instruments). A mask was fitted, the cannula and lead wires were connected, and the crocodile was placed on a treadmill belt. The crocodile was left on the stationary treadmill belt for at least 1 h to obtain resting measurements for all variables at 30°C (the effects of handling and instrumentation have previously been shown to be non-significant after 60 min) (Munns, 2000). Reductions in respiratory drive were induced by lowering T_B. The room temperature was slowly reduced over 2–3 h until the crocodile’s T_B reached 20°C. Ventilation, metabolic rate and blood gases were measured again, once the crocodile’s T_B had stabilised at 20°C for a minimum of 1 h. The room temperature was then slowly returned to 30°C and the crocodile’s T_B restabilised at 30°C for at least 1 h. Increases in centrally mediated respiratory drive were induced by short bouts of moderate-intensity exercise or by the administration of hypercapnic gas (5% CO₂). After a minimum period of 1 h at 30°C, the crocodile was exercised on the treadmill. The exercise period consisted of a two-minute exercise bout at 1.0 km h^–1. Locomotion was initiated by gently tapping the treadmill belt behind the crocodile or by lightly touching the crocodile’s tail. Following exercise, crocodiles were allowed to rest on the treadmill for a minimum of 1 h (until ventilation, blood gases and lactate concentrations had returned to pre-exercise values) and then exposed to 5% CO₂ for 10 min. The above experimental protocol was then repeated no less than 48 h after the diaphragmaticus muscle was inactivated.

All signals were collected on a computer at 1 kHz using Chart data acquisition software (AD Instruments). Because of the intermittent and variable nature of reptilian ventilation and the low breathing frequencies employed at rest, ventilatory variables were calculated from the last 10 min of the rest periods. To avoid locomotor interference on recorded signals (e.g. ventilation, EMG signals), calculations were made from the first 25 breaths immediately following exercise.

The effect of severing the diaphragmaticus muscle on all parameters was determined using a paired Dunnett’s test (30°C resting as the control, P<0.05) and paired t-tests (P<0.05). All data presented are means ± s.e.m.

Crocodiles resting at 30°C displayed a typical crocodilian breathing pattern, which consisted of one or two consecutive breaths interspersed with long pauses (Fig. 1A), V̇_E (27.61± 4.03 ml kg^–1 min^–1), V_T (15.56±3.27 ml kg^–1), f (1.98±0.48 min^–1), V̇_O2 (0.83±0.24 ml kg^–1 min^–1), V̇_CO2 (0.70±0.19 ml kg^–1 min^–1), ACR O₂ (47.00±21.06), ACR CO₂ (52.99±22.17) and RER (0.87±0.04) (Figs 2, 3, 4, 5). EMG activity from the diaphragmaticus muscle was typically associated with ventilation when crocodiles were quietly resting at 30°C (Fig. 1).

At this temperature, transection of the diaphragmaticus muscle did not induce any significant alterations in the ventilatory, respiratory or blood gas variables (Figs 6 and 7, Table 1).

A lower T_B altered the breathing pattern by increasing T_NVP and T_I (Fig. 2). Decreases in V̇_O2, V̇_CO2 (Fig. 4) and VT_I/T_I also accompanied a decrease in T_B. Diaphragmatic EMG activity was not always evident during inspiration, but when EMG activity was present, it was associated with inspiratory flow (Fig. 1B). At 20°C, transection of the diaphragmaticus muscle induced a significant increase in V_T, with no change in any other ventilatory, respiratory or blood gas parameter (Table 1).

During the immediate recovery from treadmill exercise, V̇_E increased 9-fold (Fig. 4), V_T increased 2.7-fold (Fig. 2), f increased 3.3-fold (Fig. 3), V̇_O2 increased 2.5-fold (Fig. 4) and V̇_CO2 increased 5.8-fold (Fig. 4), while blood lactate concentration rose 5.6-fold from 0.77±0.43 mmol l^–1 to 4.27±0.95 mmol l^–1 (Fig. 7). The increase in V_T was achieved via both a 1.9-fold increase in VT_I/T_I and a 1.6-fold increase in T_I (Fig. 2). While Pa_O2 remained unaltered by exercise, Pa_CO2 significantly decreased (Fig. 6).

All animals completed the exercise period both before and after inactivation of the diaphragmaticus muscle. Exercise in crocodiles with an inactivated diaphragmaticus muscle resulted in a reduction in the exercise-induced elevation in VT_I/T_I, resulting in lower V_T (Fig. 2) and V̇_E (Fig. 4) compared with the same crocodiles with intact diaphragmaticus muscles. V̇_O2 and V̇_CO2 were not significantly elevated in crocodiles with inactivated diaphragmaticus muscles (Fig. 4), and no significant alterations in blood gases were measured (Figs 6, 7).

At rest, inhalation of normoxic air with 5% CO₂ increased V̇_E 1.5-fold (Fig. 4) via a 1.5-fold increase in T_I and a 2.2-fold increase in V_T (Fig. 2). There were no significant alterations in T_NVP or f (Fig. 3) or any other ventilatory parameter (Fig. 5). EMG activity from the diaphragmaticus muscle was present during hypercapnic exposure; however, not all ventilations were associated with diaphragmaticus activity (Fig. 1). Transection of the diaphragmaticus muscle did not significantly alter any ventilatory parameter during hypercapnic exposure (Table 1).

Inactivation of the diaphragmaticus muscle in juvenile C. porosus did not induce any significant alterations in ventilation, gas exchange or arterial blood gases at 30°C, 20°C or following inhalation of 5% CO₂ (Table 1). Loss of diaphragmatic function disabled the hepatic piston pump, thus aspiration could only be achieved via alterations in intercostal or abdominal muscle activities. The resting breathing patterns of crocodiles in this study at both 20°C and 30°C, and in response to hypercapnia, were similar, both before and after surgery, to those previously measured in juvenile alligators and crocodiles under similar conditions (Farmer and Carrier, 2000c; Hartzler et al., 2006a; Munns et al., 1998; Munns et al., 2005). This suggests that the surgical intervention did not adversely alter the animals’ breathing patterns, and the consistency of ventilatory and metabolic data both before and after surgery precluded the need for sham-operated controls.

Our results suggest that activity of the inspiratory muscles (such as the intercostals, trapezius, anterior serratus and derived hypobranchial muscles of the neck) is able to maintain ventilation, thus maintaining arterial oxygenation to support metabolic rate in the absence of a functional hepatic piston pump. As such, they support the argument that the diaphragmaticus muscle is an accessory, not a primary, muscle of inspiration in crocodiles.

Variation in respiratory muscle activity of the diaphragmaticus appears to exist based on the physical environment and physiological condition of the crocodilians. It may vary in animals on land versus in water, at rest versus undergoing exercise. Earlier studies reported that intercostal muscle activity was not regular or obligatory during ventilation in submerged caiman (Gans, 1971; Gans and Clark, 1976) whereas others reported that lung ventilation can be effected solely by the use of the intercostal musculature in juvenile alligators on land (Hartzler et al., 2004; Uriona and Farmer, 2006). Uriona and Farmer also demonstrated that transection of the diaphragmaticus muscle did not alter the maximum inspiratory volume, expired volume, inspiratory or expiratory times (Uriona and Farmer, 2006). The same authors also propose that the diaphragmaticus muscle may have a limited contribution to ventilation in fasted, standing alligators. The differential role of the diaphragmatic activity in an aquatic versus terrestrial environment has been highlighted by Uriona and Farmer’s findings that the diaphragmaticus is recruited in alligators to control buoyancy and pitch during diving (Uriona and Farmer, 2008).

Some of the variation reported in activity of the diaphragmaticus and intercostal muscles may be due to the use of different sized animals in the various studies. Relatively large (up to 7.5 kg) submerged caimans were used in studies that reported low EMG activity of the intercostals and a high reliance on the diaphragmaticus muscle for inspiration (Gans, 1971; Gans and Clark, 1976). Videoradiographic studies in juvenile alligators (mass 0.72–2.09 kg) estimated that 36–61% of V_T was attributable to diaphragmaticus activity and ∼40% attributable to costosternal activity (Claessens, 2009), although it should be noted that these estimates were calculated for V_T values 2–4-fold larger than those measured at rest in this study. While the diaphragmaticus muscle is well developed in adults, it is thin and translucent in juvenile crocodilians (S.L.M., personal observations). Future investigations are needed to examine if the contribution of the diaphragmaticus muscle to ventilation increases with age in crocodilians and whether any age-related increase in diaphragmaticus muscle recruitment is related to hypertrophy of the muscle or to alterations in chest wall compliance.

Post-exercise recovery caused significant alterations in ventilatory and respiratory parameters (V̇_E, V_T, f, VT_I/T_I, T_I, V̇_O2 and V̇_CO2; Figs 2, 3, 4, 5) and arterial lactate (Fig. 7) in crocodiles with an intact diaphragmaticus muscle. The changes in ventilation and metabolic rates were not as extensive as those previously reported in exercising juvenile alligators (Farmer and Carrier, 2000b; Munns et al., 2005). The discrepancy of our results with those of earlier reports, however, is not surprising given the differences in the species used (Crocodylus versus Alligator), experimental protocol (2 min period versus exhaustive exercise) and acclimation to treadmill (none versus extensive). The aim of this experiment was not to achieve maximum treadmill performance but rather to test if adequate ventilation was maintained during elevated respiratory drive in the absence of a functional hepatic piston pump.

Transection of the diaphragmaticus resulted in a reduced capacity for exercise recovery to elevate VT_I/T_I (Fig. 2), thus limiting the elevations in V_T (–19%) and V̇_E (–39%, Fig. 4), compared with the same crocodiles with an intact diaphragmaticus. Interestingly, post-exercise-induced elevations in V_T were achieved via increases in both VT_I/T_I and T_I whereas hypercapnia-induced increases in V_T of similar magnitude were supported solely by increases in T_I. Increases in T_I reflect a delay in the centrally integrated inspiratory ‘off switch’ (Munns et al., 1998) and as such are unlikely to be altered by the transection of the diaphragmaticus muscle. However, increases in VT_I/T_I likely reflect an increase in respiratory muscle recruitment, thus increasing the rate of inspiratory airflow. Effective recruitment of the diaphragmaticus muscle to increase inspiratory airflow rates was prevented in crocodiles with inactivated hepatic piston pumps, and thus V_T and V̇_E were compromised during the recovery from exercise. VT_I/T_I was also impaired following transection of the diaphragmaticus muscle in juvenile post-prandial alligators (Uriona and Farmer, 2008), thus the proposed role of the diaphragmaticus muscle in increasing inspiratory airflow rates appears to include not only exercising but also digesting crocodilians.

Under laboratory conditions, exercise in crocodilians is predominantly anaerobic; arterial lactate concentrations increased by 5.6-fold after moderate activity in this study (Fig. 7) and by 16-fold following exhaustive exercise in alligators (Hartzler et al., 2006b). While respiratory parameters tend to increase with treadmill speed, cardiovascular responses appear to be ‘all or nothing’ with maximal increases in heart rate, central venous pressure, arterial blood pressure and venous return reached early in the exercise period, and no further elevations triggered by increasing treadmill speed (Munns et al., 2005). Exercise in crocodilians is also associated with a marked relative hyperventilation (Farmer and Carrier, 2000b; Farmer and Carrier, 2000c; Hartzler et al., 2006b), which was evident in this study by the increased ACR O₂ (Fig. 5) and the decrease in Pa_CO2 (Fig. 6). Exercising crocodiles rely on anaerobic metabolism, which results in a low demand for O₂. At the same time, a relative hyperventilation occurs during exercise and results in a high O₂ supply. The combination anaerobic metabolism (thus, low O₂ demand) and relative hyperventilation (thus, high O₂ supply) may limit the impact of the V_T and V̇_E constraints induced by transection of the diaphragmaticus muscle during exercise. Future studies involving a greater range of treadmill speeds and exercise durations would be required to more completely assess the contribution of the diaphragmaticus muscle (and hence the hepatic piston pump) to exercise endurance.

In conclusion, the contribution of the hepatic piston pump and costal ventilation, the two primary ventilatory mechanisms in crocodilians, appears to be highly plastic. In C. porosus, the diaphragmaticus muscle appears to make only limited contributions to maintaining ventilation, metabolic rate and arterial oxygenation at rest (both at preferred and lowered body temperatures) and during increased respiratory drive induced by hypercapnia. V_T elevations produced by increasing the duration of inspiration (as induced by hypercapnia) are not affected by the inactivation of the diaphragmaticus muscle. However, the diaphragmaticus muscle makes a significant contribution to ventilation during the recovery from exercise, facilitating increases in inspiratory airflow rates and thus improving the increases in V_T and V̇_E that would otherwise be obtained.

 
• ACR CO₂

  air convention requirement for CO₂

 
• ACR O₂

  air convection requirement for O₂

 
• Ca_O2

  arterial O₂ content

 
• f

  breathing frequency

 
• F_CO2

  fractional concentration of CO₂

 
• F_O2

  fractional concentration of O₂

 
• [Hb]a

  arterial haemoglobin concentration

 
• [La]a

  arterial lactate concentration

 
• Pa_CO2

  arterial blood partial pressure of CO₂

 
• Pa_O2

  arterial blood partial pressure of O₂

 
• pHa

  arterial pH

 
• RER

  respiratory exchange ratio

 
• T_B

  body temperature

 
• T_E

  expiratory duration

 
• T_I

  inspiratory duration

 
• T_NVP

  duration of non-ventilatory period

 
• V_T

  tidal volume

 
• VT_I/T_I

  rate of inspiratory airflow

 
• V̇_CO2

  rate of CO₂ consumption

 
• V̇_E

  minute ventilation

 
• V̇_O2

  rate of O₂ consumption

We are grateful to Tobie Cousipetcos and Eva Suric for assistance with crocodile care.