In vivo measurement of lung volume in ringed seals: insights from biomedical imaging

A key question in comparative physiology is how air-breathing vertebrates remain active under water for long periods on a single breath (Butler and Jones, 1997; Ponganis, 2015; Scholander, 1940). To support diving, marine mammals rely on oxygen reservoirs compartmentalized in blood, muscle and lungs. Blood and muscle oxygen stores are well studied in marine mammals relative to lungs. This can be attributed to reduced dependence on pulmonary oxygen stores in marine mammals relative to that in terrestrial species, as well as the difficulty of obtaining quantitative measurements from living, freely diving individuals (Ponganis and Williams, 2016).

Standard metrics of respiratory function include minimum air volume (MAV) and total lung capacity (TLC). MAV is the minimum volume of air in relaxed lungs (Fahlman et al., 2011; Kooyman, 1973), while TLC refers to lung volume at maximum inhalation or when manually inflated to a standard air pressure of 30 cm H₂O or 22 mmHg (Denison et al., 1971). TLC is not easily determined in living animals; for this reason, several techniques are employed to determine other respiratory parameters, which are then used to estimate TLC (Wanger et al., 2005). These methods include nitrogen washout (Sue, 2013), whole-body plethysmography (Kooyman et al., 1972; Lenfant et al., 1970) and various respirometry approaches (Scholander, 1940). In addition, allometric scaling relationships derived from empirical measurements enable estimation of TLC from body mass when species data are not available. For marine mammals, scaling relationships reported by Kooyman (1973, 1989) are used; however, these have not been updated recently, and source data for TLC often include pooled values of mixed age classes. Even so, this approach has been used ubiquitously with the assumption that estimates of TLC will hold across a wide range of body sizes and age classes.

TLC and MAV can be measured postmortem (Burns et al., 2007; Denison et al., 1971; Fahlman et al., 2011; Kooyman and Sinnett, 1979; Lydersen et al., 1992; Mitchell and Skinner, 2011) by excising the complete respiratory tract and measuring associated water displacement in both non-inflated (resting) conditions (i.e. MAV) and inflated conditions (i.e. TLC). The difference in displacement between each condition is related to the volume of the respiratory tract. Researchers interested in mammalian diving physiology rely on these postmortem estimates despite there being little information regarding the reproducibility of ex situ values in living animals (Fahlman et al., 2020b), with only a few studies directed at comparing pulmonary function and positioning both in situ and ex situ (Chevalier et al., 1978; Fahlman et al., 2014; Soutiere and Mitzner, 2004; Standaert et al., 1985).

Biomedical imaging has emerged as a valuable tool to examine comparative respiratory anatomy (Denk et al., 2020; Moore et al., 2011; Ponganis et al., 1992; Smodlaka et al., 2009), including the air reservoirs within living animals such as mice (Mitzner et al., 2001), dogs (Chevalier et al., 1978) and seabirds (Nevitt et al., 2014; Ponganis et al., 2015). Air spaces can be visualized and quantified using three-dimensional reconstructions of respiratory structures in both postmortem and living, anesthetized individuals. Importantly, this approach also allows for calculation of body volume (Ponganis et al., 2015), which can be used to evaluate body density and buoyancy.

We used computed tomography (CT) imaging data obtained during routine veterinary procedures to examine in vivo lung volume, lung capacity and whole-body buoyant force in living ringed seals (Pusa hispida). Their small body size and ease of handling enabled high-resolution volumetric quantification of discrete respiratory structures, including the anatomical dead space and individual lungs, as well as whole-body volume. We report respiratory parameters for the smallest phocid species, provide insight into the applicability of allometric scaling relationships, and discuss ecological implications of our findings for free-ranging seals.

One female and three male subadult ringed seals, Pusa hispida (Schreber 1775), were evaluated. Age was estimated from the length, mass and overall development of each individual at intake for rehabilitative care at the Alaska SeaLife Center (Seward, AK, USA). Length and mass were determined within 1 week of the CT procedure. Standard length (linear distance from nose to tail) was either directly measured on the day of the CT procedure or measured from full-body scans. Animal mass was obtained via a platform scale (W.C. Redmon Co., Peru, IN, USA; or Ohaus SD751, Ohaus Corp., Parsippany, NJ, USA). Two individuals (PH1701 and PH1804) presented with verminous pneumonia at intake and were treated with anti-helminthic drugs during rehabilitation, with resolution prior to imaging. Thus, the scans included in this study represent healthy individuals cleared of parasites, with no clinical evidence for lungworm infection present at the time of the scans.

Seals were briefly restrained at the Alaska SeaLife Center and given a pre-anesthetic intramuscular injection of midazolam (0.2–0.5 mg kg⁻¹) and butorphanol (0.24–0.7 mg kg⁻¹) (see Woodie et al., 2020). Following sedation, a single lumen central venous catheter (16–18 g, 13–15 cm) was placed in the epidural vertebral sinus flushed with heparinized saline and capped as in Goertz et al. (2008). Patency of the soft catheter was ensured prior to transport to the nearby imaging facility. Prior to the CT procedure, propofol (2–3 mg kg⁻¹) was administered intravenously via the catheter to allow for intubation and inflation of an endotracheal tube cuff. Seals were maintained on oxygen and isoflurane gas for the duration of the procedure. Full inflation of the cuff prevented air leakage around the tube. Supplemental intravenous propofol was titrated incrementally to facilitate intentional apneic intervals during scanning with manual, intermittent, positive-pressure ventilation prior to and following each imaging series. A non-steroidal anti-inflammatory medication (meloxicam, 0.2–0.5 mg kg⁻¹), and broad-spectrum antibiotic (cefazolin, 10–20 mg kg⁻¹) were administered intravenously via the catheter. Following the CT procedure, sedation was reversed with separate injections of intramuscular or intravenous naltrexone (2 mg naltrexone per 1 mg butorphanol) and intravenous flumazenil (1 mg flumazenil per 20 mg midazolam). The endotracheal tube was removed after regular spontaneous respirations resumed. Following extubation, seals were returned to the Alaska SeaLife Center where they resumed normal eating and activity within an hour. The duration of anesthesia was less than 1 h from propofol induction to recovery and extubation.

CT scans were performed with a GE 16 Light Speed Scanner, GE 16 Bright Speed Scanner (General Electric Healthcare, Chalfont St Giles, Bucks, UK), or a Siemens 32/64 Somatom GO-UP Scanner (Siemens, Munich, Germany). Modified thorax protocols (Table S1; S.D.-G., unpublished) were used to obtain optimized images of the full respiratory tract with slice thickness of 0.625–2.5 mm. An initial scan was obtained on two seals (PH1802 and PH1804) in sternal recumbency without lung inflation during apnea, with the pressure gauge of the anesthesia circuit at 0 mmHg. This condition was defined as the resting, relaxed position of the lungs when the seal was out of water. All seals were scanned in sternal recumbency with lungs hyperinflated to a pressure of 30 mmHg. To test for replicability of lung volume at a given pressure, variation in volume within inflation conditions, and the difference in volume as a result of patient position, one seal (PH1802) received additional scans in both dorsal and sternal recumbency at inflation pressures of both 30 and 37 mmHg.

Animal handling activities including rescue, rehabilitation and diagnostic CT procedures were authorized under NOAA's Marine Mammal Health and Stranding Response/Research Program 18786, Stranding Agreement SA-AKR-2019-01, and marine mammal research permit 18902. Research was approved by the Institutional Animal Care and Use Committees at the University of California Santa Cruz and the Alaska SeaLife Center.

To determine key respiratory parameters at known lung inflation conditions, DICOM images from CT series were imported to 3D Slicer (Fedorov et al., 2012; https://www.slicer.org/) and converted into closed-surface three-dimensional models. Anatomical structures were manually separated into volumetric segmentations of trachea, bronchi, and left and right lungs based on tissue attenuation (Fig. 1). Tracheal volume was defined from the image immediately caudal to the laryngeal cartilages extending to the image of the cranial margin of the carina. Bronchial volume was defined as the region from the carina to distal portions of the cranial and caudal lobar bronchi. Bronchioles were too diffuse to manually trace, so their volume is included in the volume of the lungs. TLC included both the tissue and air spaces of the left and right lungs, in addition to the bronchioles. The inclusion of the tissue and air spaces is in line with other studies (Lydersen et al., 1992) with which we compared our values. The volume of each segment was calculated in cubic centimeters and converted to milliliters.

Segments of the respiratory tract were considered with respect to whether surfaces were available for oxygen exchange. Anatomical dead space (the portion not in contact with gas exchange surfaces) was defined as the volume of the trachea plus the volume of the bronchi; this measure is not equivalent to respiratory dead space (Fowler, 1948; Rossier and Bühlmann, 1955) as the bronchioles could not be partitioned from the tissues of the lungs in this study. MAV was characterized here as the lung volume in the non-inflated (resting) condition, with inflation pressure of 0 mmHg. Because individuals were measured out of water, we presume this metric will differ somewhat from MAV values obtained from seals resting in water at the surface (Fahlman et al., 2020a). TLC was determined as the volume of the inflated lungs at 30 mmHg. This pressure is higher than the standard of 22 mmHg (30 cm H₂O) used to measure TLC in other mammalian studies (Denison and Kooyman, 1973; Denison et al., 1971; Kooyman and Sinnett, 1982; Loring et al., 2016; Moore et al., 2011; Weibel, 1973), but was necessary for the clinical diagnostic protocol. Specifically, the ringed seals’ lungs were hyperinflated to ensure that no atelectasis (partial or full collapse) or scarring of lung tissue was present. To obtain the proportion of blubber that contributed to total body volume, blubber was segmented and quantified for the two animals for which we had whole-body scans (PH1802 and PH1804). Further, surface area to volume ratio (SA:V) was directly measured for seal PH1802.

To compare our results with those of other marine mammals, we considered the commonly used allometric scaling equation: TLC=0.1M_b^0.96 (Kooyman, 1989) (where M_b is body mass). We also determined another scaling equation specific to pinnipeds. The TLC data included in the pinniped-only allometric plot either were collected empirically or could be calculated from empirically reported mass-specific total lung oxygen stores. We evaluated our primary measure of mean TLC as a function of mean total body mass for the subadult ringed seals in our study. We then compared our results with expected values from these allometric relationships to determine whether total body mass is a reliable indicator of total lung capacity for ringed seals.

Primary comparisons of respiratory structures and volumetric analyses were made at lung inflation pressures of 0 and 30 mmHg in sternal recumbency (Table 1). When inflated to 30 mmHg, TLC ranged from 870 to 2271 ml, resulting in mass-specific values between 52 and 92 ml kg⁻¹. The right lung was larger than the left in all individuals in the inflated condition, with an average size difference of 6.3%. For two individuals measured in the non-inflated condition, MAV was 564 and 886 ml, with mass-specific values of 22 and 32 ml kg⁻¹. Lung volume for these individuals increased by a factor of 2.5 when fully inflated.

Maximum respiratory tract volume was 904–2323 ml. This was equivalent to 11% and 18% of total body volume for the two seals with full-body scans (PH1804 and PH1802). The anatomical dead portion of the respiratory tract changed little with inflation for two individuals with comparable non-inflated and inflated scans (PH1503 and PH1802). These seals exhibited similar increases in tracheal volume (∼15%) and negligible increases in bronchi volume (∼1%) from non-inflated to inflated conditions. Thus, while the volume of the total respiratory tract changed by an average factor of 2.5 when the lungs were inflated, most of this difference was due to changes in lung volume.

Replicate scans in sternal recumbency at 37 mmHg for seal PH1802 showed that TLC varied by 5% (74 ml) between scans. Lung volume varied similarly between 30 and 37 mmHg, with an increase of 5% (75 ml) at the higher inflation pressure. When hyperinflated to 37 mmHg, lung volume was 13% (194 ml) greater in dorsal recumbency than in sternal recumbency.

Full-body scans for seal PH1802 were evaluated in both inflated and non-inflated conditions to determine changes in respiratory tract volume and total body volume. The increase in respiratory tract volume was 815 ml. In contrast, total body volume in the inflated condition increased by only 446 ml, equivalent to a 2% increase in body volume. The directly measured surface area of this seal was 58,193 cm² and its total body volume was 25,004 cm³, resulting in a SA:V of 2.3:1. For the two seals for which total blubber volume could be measured from CT scans, blubber by total body volume was 33% (PH1804) and 49% (PH1802).

The scaling equation we determined for pinnipeds using previously published values (TLC=0.1M_b^0.98) is remarkably close to the classic relationship reported by Kooyman (1989) for marine mammals (Fig. 2). Indeed, the Kooyman (1989) equation falls within the 95% confidence interval of the pinniped-only equation, suggesting that this offset is not significant. Source data for the pinniped-only relationship are provided in Table S2 (Burns et al., 2007; Falke et al., 2008; Kooyman and Sinnett, 1982; Lenfant et al., 1970; Lydersen et al., 1992; Reed et al., 1994). The ringed seals in this study are the smallest pinnipeds for which TLC data are now available. When compared with the scaling relationships described above, our in vivo measurements obtained from ringed seals are about 27% lower than predicted.

Body density and buoyant force were calculated from the measured total body volume of one individual (PH1804) in the inflated condition, and another individual (PH1802) in both non-inflated and inflated conditions. Taking body mass into account, both seals exhibited similar body density irrespective of inflation condition. Individual PH1802 was denser than seawater (1.027 g ml⁻¹ at 10°C) at both 0 mmHg (1.052 g ml⁻¹) and 30 mmHg (1.033 g ml⁻¹) inflation conditions, whereas seal PH1804 was less dense than seawater (0.989 g ml⁻¹) in the inflated condition. Based on these measurements PH1802 had negative net vertical forces in both non-inflated (−6.0 N) and inflated (−1.3 N) conditions. In contrast, PH1804 had a positive net force of 7.8 N in the inflated condition.

The anatomical dead space of ringed seals comprised only 3% of total respiratory tract volume and changed little between non-inflated and inflated conditions. This negligible change can be attributed to the rigid hyaline cartilage reinforcement of the trachea (Smodlaka et al., 2009), which aids in lung collapse while diving by allowing compressed air from the lungs to be stored within this non-compliant compartment (Kooyman, 1973). The largest volume measured of the air-filled respiratory tract – including dead space and TLC – was 2.3 l. We found that TLC was three times smaller in our ringed seals measured in vivo than in ringed seal lungs assessed postmortem (Lydersen et al., 1992). This could be due in part to constraints of lung inflation within an enclosed body cavity versus when the respiratory tract is excised. While developmental differences may confound comparisons across age classes, our mass-specific estimates of TLC were also smaller than measures obtained from the excised lungs of adult seals (Lydersen et al., 1992), suggesting that postmortem measurements may overestimate lung capacity.

Given the hyperinflation applied during prescribed veterinary assessments, our measurements provide an upper bound of TLC. Notably, we found little difference in TLC at pressures of 30 and 37 mmHg, indicating that the lungs reached maximum expansion in both conditions. While normally measured at a standard pressure of 22 mmHg, the TLC values reported are likely biologically relevant as they capture full inflation of the lungs within the body cavity; however, they may not be physiologically accurate as a result of hyperinflation. We found that subject positioning had a greater influence on lung volume than inflation pressure, highlighting the differential effects of gravity and recumbency on TLC estimates obtained out of water. Measurements conducted in dorsal recumbency allowed for more complete expansion of the lungs and chest wall and more accurate assessment of TLC.

We measured MAV that was about 40% of TLC. This is much higher than values based on excised respiratory tracts in other marine mammals, which indicate MAV is 0–16% of TLC (Fahlman et al., 2011; Kooyman and Sinnett, 1979). Although it is a common metric, MAV can be difficult to compare across studies. Here, MAV was measured in living, apneic seals when lungs were relaxed in the non-inflated condition. Other studies have defined MAV as the volume of relaxed lungs when transpulmonary pressure is zero (Kooyman and Sinnett, 1979), a condition that can only be achieved postmortem. MAV has also been related to both functional residual capacity (FRC, the air volume remaining after a passive exhalation) and residual volume (RV, the air volume remaining after forceful exhalation) in living animals (Fahlman et al., 2011). Our definition of MAV most closely aligns with FRC; therefore, comparisons to postmortem studies of other marine mammals may not be appropriate.

Diving lung volume (DLV) is commonly estimated at 50% of TLC for pinnipeds, with an oxygen extraction efficiency of 15% (Kooyman, 1973; Kooyman and Sinnett, 1982; Kooyman et al., 1971). As direct measurements exist for only a few species (Kooyman et al., 1971; Ponganis, 2011), we often rely on these assumptions to quantify mass-specific DLV. For our ringed seals, the traditional assumptions yield a DLV ranging from 0.4 to 1.1 liters and corresponding mass-specific DLV from 3.9 to 6.9 ml kg⁻¹. Similar to TLC, these values for DLV in immature ringed seals are lower than previously reported for adult ringed seals (Lydersen et al., 1992), and more similar to values reported for harbor seal pups (Burns et al., 2005). Although the assumptions outlined above can be useful in estimating DLV when empirical data are lacking, much remains to be learned about how respiratory capacity including DLV may change across ontogeny.

Relative to predictions based on scaling relationships, the immature ringed seals in this study had lower than expected lung capacity. This was also the case for adult ringed seals measured postmortem (Lydersen et al., 1992), suggesting the relatively small TLC values obtained here are not explained by methodology or ontogeny. Rather, the deviation of ringed seal lungs from common scaling relationships may be explained by their compact body size and extensive blubber stores. One of the novel aspects of this work was our ability to directly measure SA:V in one individual. This metric is rarely empirically determined but is relevant to aspects of thermoregulation, hydrodynamics and energetics. To compensate for large SA:V and associated heat loss in polar waters, ringed seals have considerable blubber reserves that may comprise half their body volume. Although serving different primary functions, the relative volume of both lungs and blubber have important effects on buoyancy in the smallest phocid.

Seals must manage dynamic buoyant forces and associated energetic costs while diving (Watanabe et al., 2006; Williams et al., 2000). The imaging approach employed here enabled a variety of volumetric measurements relevant to evaluating constraints on diving. Despite high blubber content (48% of body volume), seal PH1802 had a net negative (sinking) force in both inflated and non-inflated lung conditions. In contrast, seal PH1804 had lower blubber volume (33% of body volume), but exhibited a net positive (buoyant) force in the inflated lung condition. These somewhat surprising results were driven by relatively small differences in overall body size (mass and volume) and body density, although in absolute terms, both seals were almost neutrally buoyant in seawater. For reference, some penguins have a net positive force of +15 to 50 N (Ponganis et al., 2015), while larger seal species exhibit net negative forces from −15 to −132 N (Beck et al., 2000; Webb et al., 1998). In comparison, the net vertical forces on the ringed seals were relatively small (−6 to +7 N) and could likely be adjusted by changes in lung volume at the start of a dive. These near-neutral values are physiologically advantageous as they should limit the overall cost of foraging, diving and moving through the marine environment (Adachi et al., 2014; Miller et al., 2012; Nousek-McGregor et al., 2014; Richard et al., 2014; Sato et al., 2013).

We conclude that in vivo measurements of lung capacity in ringed seals are smaller in both absolute and mass-specific terms relative to postmortem assessments. Further, total body mass consistently underestimates TLC in this species when considered in the context of established allometric relationships. This deviation likely results from their small, compact body size and exceptional blubber stores. Biomedical imaging can provide accurate quantification of specific regions of the respiratory tract, as well as additional measures of total body and blubber volume that have important ecological implications for free-ranging individuals.

We thank the stranding response and animal care teams at the Alaska SeaLife Center, as well as D. Rosen, C. Goertz, J. Belovarac, K. Strehlow, P. Martre, R. Cieri, J. Rasmussen, R. Nichols, R. Sills, T. Williams and A. Moore.