In 1940, Per Scholander (1940) published an article that described the unusual properties of marine mammal respiratory systems. Scholander suggested that the highly compliant lung and rib cage would easily compress, the air being shunted into the rigid upper airway. This hypothesis has remained a central tenet in marine mammal diving physiology, with the assumption that this physiological trait will reduce uptake of N₂ and the risk of decompression sickness (DCS). A few studies have attempted to determine how pressure affects gas exchange in forced or freely diving marine mammals. These studies have assessed alveolar compression either directly by quantifying the depth related pulmonary shunt (Kooyman and Sinnett, 1982; McDonald and Ponganis, 2012), or indirectly by measuring N₂ uptake in arterial and venous blood during a dive (Kooyman et al., 1972; Falke et al., 1985) or measuring N₂ removal from the muscle following a series of repeated dives (Ridgway and Howard, 1979; Houser et al., 2010). As the estimated depth at which alveolar collapse occurs differs considerably between studies, Bostrom et al. (2008) developed a mathematical model in an attempt to understand lung compression in marine mammals. The objective was to create a theoretical frame-work that could be used to predict the air volumes of the various compartments of the respiratory system to the limit of collapse. The results from the model suggested that the diving lung volume, the relative size of the upper (conducting airways) and lower (alveoli) airways, and the structural properties (compliance) of the upper and lower airways were important in determining the depth at which the alveoli collapse and gas exchange ceases (Bostrom et al., 2008). The model output was compared to available data for collapse depth in different species (Kooyman et al., 1970, 1972; Ridgway and Howard, 1979; Kooyman and Sinnett, 1982; Falke et al., 1985), and it was concluded that behavioral (diving lung volume) and structural (lung and dead space compliance) variations between species could account for the observed species differences (Bostrom et al., 2008; Fahlman et al., 2009).

Static pressure-volume loops are commonly used to measure the physical properties (compliance) of the respiratory system. Published data exist on excised lungs for several terrestrial species, but only a few measurements have been made for marine mammals (Denison et al., 1971; Piscitelli et al., 2010; Fahlman et al., 2011; Moore et al., 2014). A recently published theoretical study compared estimated blood and tissue gas tensions (O₂, CO₂, and N₂) using species-specific respiratory compliance estimates or those previously published from terrestrial mammals (Hodanbosi et al., 2016). The results showed that the blood and tissue PN₂ levels were significantly lower using the species-specific compliance estimates, while there were little or no differences for blood and tissue PCO₂ or PO₂ levels. It is therefore difficult to assess the accuracy of the model output without species-specific respiratory compliance estimates. In addition, common indices of respiratory capacity and mechanics, e.g., lung compliance, inspiratory and expiratory tidal volume, flow-rate, and duration, exist for a limited number of marine mammals of different age classes (Parker, 1922, 1932; Scholander and Irving, 1941; Robin et al., 1963; Olsen et al., 1969; Kooyman et al., 1971; Leith, 1976; Gallivan, 1981; Kooyman and Cornell, 1981; Gallivan et al., 1986; Bergey and Baier, 1987; Reed et al., 1994, 2000; Fahlman et al., 2015). Only by acquiring comparable data from multiple species, can one begin to understand the level of confidence with which one can predict such parameters for species where we have little or no data. The present study was undertaken to extend the current knowledge of the respiratory physiology and mechanical properties of Patagonia sea lions (Otaria flavescens) participating voluntarily. In addition, we also collected end-expiratory gases and estimated breath-by-breath gas exchange. Our results indicate that the metabolic demand and respiratory physiology of Patagonia sea lions are similar to other pinniped species.

A total of 30 experimental sessions were conducted in May and October 2015. The sex, age, and average during the measurement are reported in Table 1.

This is the first study having measured respiratory physiology and metabolic rate in Patagonia sea lions during voluntary breathing. Physiological studies on trained animals that participate voluntarily are useful as they minimize the stress involved as compared with studies using forced restraint. The results suggest that the sC_L is similar to those measured in anesthetized sea lions, and significantly higher as compared with humans (Fahlman et al., 2014a). In addition, the measured tidal volumes during voluntary breaths are higher than those predicted in terrestrial mammals, and the respiratory flow similar to those reported in California sea lions (Kerem et al., 1975; Matthews, 1977). The estimated resting metabolic rates were similar to those measured in Steller sea lions and California sea lions in water (Hurley and Costa, 2001; Fahlman et al., 2008, 2013) and Steller sea lions in air (Rosen and Trites, 2000). Although the mass specific metabolic rate appeared similar between females and the single male sea lion, data from additional males are required to address this question meaningfully. Thus, the results presented here are likely to represent baseline metabolic and lung function values for healthy Patagonia sea lions.

Previous studies have measured respiratory physiology in a number of marine mammals, ranging from work on restrained, semi-restrained, and trained animals participating voluntarily (Irving et al., 1941; Scholander and Irving, 1941; Olsen et al., 1969; Kooyman et al., 1971; Kerem et al., 1975; Matthews, 1977; Gallivan and Best, 1980; Gallivan, 1981; Kooyman and Cornell, 1981; Reed et al., 1994, 2000; Fahlman et al., 2015). An important aspect for studies on trained animals is that the individual subject participates voluntarily. This helps minimize the potential stress associated with restraint, and exposure to novel environments, or situations. However, for studies on trained animals the influence of the trainer cannot be discarded. For this reason, it is important that the study subjects undergo desensitization to assure that the experiment minimally affect the physiological variables being measured. In the current study, the respiratory frequency during the research trials was compared with focal observations of each animal while they were undisturbed. These data confirm that the experimental procedure did not alter the breathing frequency of the animals. Another important aspect to consider is that the measurements were made in sternal recumbancy. There may be differences in respiratory function and mechanics in this position as compared to animals in water, due to the compression on the chest. Thus, this is an important aspect to investigate in future studies.

The measured tidal volumes increased with size (Figure 3), and following correction the mass-specific tidal volume ranged from 47 to 73% of TLC_est. This mass-specific tidal volume is significantly higher as compared with the estimated value of 14% in terrestrial mammals (Stahl, 1967). Mortola and Sequin (2009) assembled published data on VT for marine mammals and the equation predicting tidal volume was VT = 22 Mb1.01, where VT is tidal volume in mL, and M_b the body mass in kg. The results in the current study agree well with the tidal volume estimates from this equation, and are consistent with the general observation by others that marine mammal take deeper breaths (Olsen et al., 1969; Kooyman, 1973; Matthews, 1977; Kooyman and Cornell, 1981; Fahlman et al., 2015) and have a reduced breathing frequency (Mortola and Limoges, 2006) as compared with terrestrial mammals. The marine mammal breathing strategy seems to be conserved evolutionary, expressed at birth, and maintained in both fully aquatic (cetaceans) and semi-aquatic (pinnipeds) marine mammals (Mortola and Limoges, 2006). It has been suggested that increased buoyancy at the water surface may be one possible explanation for this breathing strategy (Mortola and Limoges, 2006). There is human clinical interest in this breathing pattern, called airway pressure release ventilation (APRV), as it may be useful to improve gas exchange during anesthesia (Daoud et al., 2012). Thus, this breathing pattern may help prevent a substantial increase in alveolar CO₂ (P_ACO₂) (Mortola and Sequin, 2009) during intermittent/apneustic breathing. Breath-by-breath respirometry provides a useful tool to study the possible evolutionary advantages of the aquatic breathing strategy, with an extended inspiratory pause, as compared with terrestrial breathing pattern, and may improve knowledge how marine mammals manage gases during diving.

Breath-by-breath respirometry provides additional advantages from conventional flow-through respirometry, but is also logistically challenging. For example, the O₂ uptake and CO₂ produced can be estimated for each breath, making it possible to determine how blood gases change following exercise (Fahlman et al., 2016), or diving (Kooyman et al., 1973; Reed et al., 1994, 2000). To estimate the O₂ taken up on a breath-by-breath basis requires matching of respiratory flow and expired gas content. This is challenging if the respiratory flow is fast and the breath duration short. In a previous study, we managed to reliably estimate O₂ uptake and CO₂ production per breath in bottlenose dolphins (Fahlman et al., 2015). As the breath duration is 2–4 times as long (Figure 2), and the respiratory flow an order of magnitude slower (Figure 4) in the sea lions as compared with the dolphins, the O₂ uptake should be accurately estimated. In addition, the estimated mass-specific resting metabolic rates (sRMR: 4.1–10.5 mL O₂ min⁻¹ kg⁻¹) were similar to those measured in Steller sea lions and California sea lions in water (Steller sea lions: 7.4–9.2 mL O₂ min⁻¹ kg⁻¹; California sea lions: 5.7–10.4 mL O₂ min⁻¹ kg⁻¹, Hurley and Costa, 2001; Fahlman et al., 2008, 2013) and Steller sea lions in air (3.0–9.5 mL O₂ min⁻¹ kg⁻¹, Rosen and Trites, 2000). As both the measured tidal volumes and metabolic rates are within the range of those measured in other pinnipeds of similar size, the reported data likely represent valid estimates for this species. Thus, using this system of gas analyzer and pneumotachometer we can accurately estimate breath-by-breath O₂ uptake. In addition, the fast response gas analyzer allows us to estimate end-expiratory gas content, or end-tidal gas composition. This may provide for interesting insights into respiratory health, and be a useful non-invasive method to assess lung disease in marine mammals (Van Elk et al., 2001). Despite its challenges, breath-by-breath respirometry provides data that have wide applicability for many research fields, and has previously been used to answer interesting questions in marine mammal eco-physiology (Reed et al., 1994, 2000). Improved understanding of the physiological limitations of marine species is vital to make accurate predictions how they may respond to changes in the environment. This will further enhance conservation and management efforts to reduce the impact on marine species.

During diving marine vertebrates incur an O₂ debt that is paid back at the end of the dive (Scholander, 1940). When marine mammals dive repeatedly with only a short surface interval (a diving bout), data indicate that the O₂ debt is not repaid between dives, but that they dive again before they completely replenish the O₂ stores (Fahlman et al., 2008). The rate of gas exchange is governed by the partial pressure gradient, and this is largest when the animal returns to the surface to breathe. In addition, as the O₂ dissociation curve is sigmoidal the most efficient uptake is on the steep portion of the curve. As the partial pressure gradient decreases with each breath, the amount of O₂ taken up therefore decreases with time. Thus, by not completely restoring the O₂ the animals minimize time spent at the surface and maximize time at depth (Kramer, 1988; Thompson and Fedak, 2001). However, this diving strategy is likely to cause gradual accumulation of CO₂ and/or N₂ with each subsequent dive, eventually forcing a prolonged surface interval or a period of very short and shallow dives (Scholander, 1940; Fahlman et al., 2009; Hooker et al., 2009). As a result, the management of O₂ may govern the duration of each dive and surface interval, while accumulation of CO₂ and/or N₂ may determine the duration of a bout. Changes in dive behavior, as may occur with changes in prey availability and/or environmental change may alter the accumulation of CO₂ and N₂, and thereby the foraging efficiency. Breath-by-breath analysis of gas exchange is a powerful tool to further understand how marine mammals manage gases during diving and has provided interesting results that provide information about what physiological variables that may limit diving (Reed et al., 1994, 2000; Boutilier et al., 2001). Studies using such methods would be useful to assess how changes in dive behavior and physiology may cause animals to exceed physiological limits that may cause gas bubble disease (Bostrom et al., 2008; Fahlman et al., 2009, 2014b; Fitz-Clarke, 2009; Hooker et al., 2009).

Scholander suggested that high chest and alveolar compliance, and low compliance of the conducting airways should help to minimize gas exchange during diving and thereby the risk of decompression sickness (Scholander, 1940). Denison et al. (1971) showed that the cartilaginous reinforcement of the conducting airways helped the alveoli to empty more completely in California sea lions as compared with the dog. This higher sCL as compared with terrestrial mammals have also been reported in a range of other marine mammals (Olsen et al., 1969; Leith, 1976; Bergey and Baier, 1987; Fahlman et al., 2014a, 2015) and the data in the Patagonia sea lion agree. The increased understanding of the mechanical properties of the respiratory system may help us better understand how marine mammals manage gases during diving, avoid pressure related problems, and provide clues how they perform extended dives to great depths. Modeling efforts may help increase the understanding how marine mammals respond to environmental change, but model results are affected by the quality of the data and the accuracy of the model assumptions. For example, a number of studies have attempted to model how the interaction between behavior, physiology, and environmental pressure affect the risk of decompression sickness during breath-hold diving (Houser et al., 2001; Zimmer and Tyack, 2007; Fahlman et al., 2009, 2014b; Fitz-Clarke, 2009; Hooker et al., 2009). The theoretical work performed on marine mammals used parameter estimates from terrestrial species or from marine mammals species that may be distantly related, i.e., Weddell seal vs. beaked whale. In a recent study it was shown that when modeling gas dynamics in California sea lions using species-specific parameters, the model output was significantly different as to when using parameters from terrestrial species (Hodanbosi et al., 2016). This highlights the importance of studies to better understand physiological limitations that may impact how environmental change limit survival of marine mammals.

The esophageal balloon catheter provides information of the mechanical properties of respiration (Fahlman et al., 2015). For example, in Figure 1B there was little or no change in the transpulmonary pressure during exhalation, suggesting that this phase is passive and mostly governed by the elastic recoil of the lung and chest down to FRC. At the beginning of the inhalation the transpulmonary pressure decreases which indicates that the respiratory muscles help with the inspiratory phase. Similar results were seen during voluntary breaths in bottlenose dolphins (Fahlman et al., 2015). However, in the bottlenose dolphin both the expiratory and inspiratory phases were active during maximal efforts. We currently do not have any result from sea lions performing maximal respiratory efforts but it is likely that in sea lions the respiratory muscles may help during exhalation in this species too.

AF conceived of the study, designed the experiments, collected and analyzed the data, carried out the statistical analysis, and drafted the paper; JM participated in the design of the study and was in charge of animal training.

Funding for this project was provided by the Office of Naval Research (ONR YIP Award # N-000141410563) and by Dolphin Adventure and Oceanografic Foundation.