ABSTRACT

In the present study, we examined lung function in healthy resting adult (born in 2003) Pacific walruses (Odobenus rosmarus divergens) by measuring respiratory flow () using a custom-made pneumotachometer. Three female walruses (670–1025 kg) voluntarily participated in spirometry trials while spontaneously breathing on land (sitting and lying down in sternal recumbency) and floating in water. While sitting, two walruses performed active respiratory efforts, and one animal participated in lung compliance measurements. For spontaneous breaths, was lower when walruses were lying down (e.g. expiration: 7.1±1.2 l s−1) as compared with in water (9.9±1.4 l s−1), while tidal volume (VT, 11.5±4.6 l), breath duration (4.6±1.4 s) and respiratory frequency (7.6±2.2 breaths min−1) remained the same. The measured VT and specific dynamic lung compliance (0.32±0.07 cmH2O−1) for spontaneous breaths were higher than those estimated for similarly sized terrestrial mammals. VT increased with body mass (allometric mass-exponent=1.29) and ranged from 3% to 43% of the estimated total lung capacity (TLCest) for spontaneous breaths. When normalized for TLCest, the maximal expiratory (exp) was higher than that estimated in phocids, but lower than that reported in cetaceans and the California sea lion. exp was maintained over all lung volumes during spontaneous and active respiratory manoeuvres. We conclude that location (water or land) affects lung function in the walrus and should be considered when studying respiratory physiology in semi-aquatic marine mammals.

INTRODUCTION

Marine mammals have to balance available O2 while using aerobic metabolism during foraging dives and are required to return to the surface to replenish their O2 stores and remove CO2. This necessitates efficient gas exchange to rapidly replenish the consumed O2 before commencing the next dive. The functional and mechanical adaptations of the respiratory system are crucial for gas exchange, and the scientific effort to describe and better understand these features in marine mammals has progressively increased since the first works of Irving (1939) and Scholander (1940). Past work has highlighted the anatomical and functional differences when comparing the respiratory system of marine mammals with that of their terrestrial counterparts (for reviews, see Fahlman et al., 2017; Kooyman, 1973; Piscitelli et al., 2013). For example, studies on the respiratory function in both pinnipeds and cetaceans showed that the tidal volume (VT, l) and respiratory frequency (fR, breaths min−1) are, respectively, higher and lower in marine as compared with terrestrial mammals (Fahlman et al., 2017). Also, these studies revealed that, unlike terrestrial mammals, marine mammals are able to generate high respiratory flow (, l s−1) during breaths of short duration (Fahlman et al., 2017). In addition, the structure and function of the respiratory system in marine mammals is important in the determination of their diving limitations (Bostrom et al., 2008; Fahlman et al., 2009), and increasing environmental impacts of the ocean (e.g. decreased prey availability or underwater sonar) could lead to an alteration in their diving behaviour. Therefore, an increased understanding of respiratory function in different species is important to gain a better knowledge of the physiological limitations on these species.

The respiratory physiology of the walrus (Odobenus rosmarus) has been poorly investigated and data only exist on fR in wild Atlantic walruses (Odobenus rosmarus rosmarus: Bertelsen et al., 2006; Stirling and Sjare, 1988), or fR and end-expired alveolar CO2 on one animal under professional care (Mortola and Limoges, 2006; Mortola and Seguin, 2009). Spirometry is a minimally invasive method that provides knowledge on basic respiratory function and mechanics (Burki, 1981; Crapo, 1994), and has recently been adapted for use in voluntarily participating pinnipeds and cetaceans (Fahlman et al., 2020a,b; Fahlman et al., 2019a,b, 2015; Fahlman and Madigan, 2016; Kooyman and Cornell, 1981; Matthews, 1977; Reed et al., 2000). In the present study, we aimed to increase basic respiratory physiology data in the walrus by measuring lung function and mechanics in voluntarily participating adult females using spirometry. A previous study that investigated the California sea lion (Zalophus californianus) suggested that the location (resting in water or on land) could affect respiratory function as a result of pressure on the chest (Fahlman et al., 2020b). Thus, we tested the hypothesis that body position on land (sitting or lying down in sternal recumbency) or floating in water would significantly alter lung function variables [e.g. VT, fR, total breath duration (Ttot, s) and ]. We also hypothesized that expiratory (exp) is maintained over most of the vital capacity (VC, l) in the walrus, and that lung compliance (CL, l cmH2O−1) is higher as compared with that of terrestrial mammals, as previously reported for other marine mammal species (Fahlman et al., 2017).

Our results show that in the walrus, increases when floating in water, while VT, Ttot and fR remain the same in both media. , VT, Ttot and dynamic CL were similar to those reported for other pinnipeds, where VT increased with body mass (Mb, kg) with an allometric mass-exponent close to 1. The flow–volume relationship showed a nearly constant exp over all lung volumes while performing spontaneous and active breaths. In addition, when normalized for the estimated total lung capacity (TLCest, l), exp from active respiratory manoeuvres was higher than that seen in other pinnipeds, but lower than that reported in cetaceans and in the California sea lion.

List of symbols and abbreviations

     
  • CL

    lung compliance

  •  
  • fR

    respiratory frequency

  •  
  • Mb

    body mass

  •  
  • MAV

    minimum air volume

  •  
  • Pamb

    ambient atmospheric pressure

  •  
  • Pao

    airway opening pressure

  •  
  • Poeso

    oesophageal pressure

  •  
  • PL

    transpulmonary pressure

  •  
  • sCL

    specific lung compliance

  •  
  • s

    specific respiratory flow

  •  
  • sE

    mass-specific respiratory minute volume

  •  
  • sexp

    specific expiratory flow

  •  
  • sinsp

    specific inspiratory flow

  •  
  • sVT

    mass-specific tidal volume

  •  
  • Texp

    expiratory duration

  •  
  • Tinsp

    inspiratory duration

  •  
  • Ttot

    total breath duration

  •  
  • TLC

    total lung capacity

  •  
  • TLCest

    estimated total lung capacity

  •  
  • VC

    vital capacity

  •  
  • VCB

    behavioural vital capacity

  •  
  • respiratory flow

  •  
  • E

    respiratory minute volume

  •  
  • exp

    expiratory flow

  •  
  • insp

    inspiratory flow

  •  
  • VT

    tidal volume

  •  
  • VT,exp

    expiratory tidal volume

  •  
  • VT,insp

    inspiratory tidal volume

MATERIALS AND METHODS

Study subjects

Three adult Pacific female walruses [Odobenus rosmarus divergens (Illiger 1815)], born in 2003 and housed under professional care at the Oceanogràfic (Valencia, Spain), participated in the present study (Table 1). The animals were rescued in the wild as orphan pups and were brought into the aquarium. The health of the walruses was assessed daily, and no pulmonary disease was detected during the data collection period.

Table 1.

Trial and sample details, morphometrics and respiratory frequency of Pacific walruses

Trial and sample details, morphometrics and respiratory frequency of Pacific walruses
Trial and sample details, morphometrics and respiratory frequency of Pacific walruses

Procedures, morphometrics and environmental parameters

The walruses were desensitized to the equipment and trained to perform the experimental procedures using operant conditioning. Therefore, participation in each research trial was voluntary, where the animals could end the experimental trial at any time. This procedure allowed for data collection in a relaxed physiological state. Spirometry trials while spontaneously breathing were performed while inactive in three different body positions: (1) lying down in sternal recumbency, (2) sitting while supported by the pectoral flippers and (3) floating on the water surface (see fig. 1 in Fahlman et al., 2020b). The walruses adopted a vertical position while floating in a 3 m deep seawater pool. The estimated water height acting on the centroid of the lungs was approximately 40 cm. Two of the walruses were trained to perform 5–10 consecutive maximal respiratory efforts while sitting on land. The animals performed these manoeuvres at their individual capacity and we will refer to them as active respiratory efforts/manoeuvres, where the maximum measured VT was considered as the behavioural vital capacity (VCB, l). In addition, one walrus was trained to swallow an oesophageal balloon catheter that allowed measurement of the dynamic CL during spontaneous breaths while sitting.

Data were collected from a total of 120 trials performed from February 2015 to July 2018 (2015: n=25, 2016: n=1, 2017: n=52, 2018: n=42), and a subset of 5 trials for each animal and body position (15 trials per position, n=45) were selected for the lung function analysis. Only trials where the walruses were resting for at least 2 min while performing complete breaths (composed of an exhalation and an inhalation) were included in the analysis. In addition, a total of 9 separate trials while sitting included active respiratory manoeuvres, and 3 separate trials measured dynamic CL. The Mb of each animal was recorded in the same week as lung function testing, and the mean (±s.d.) for selected trials was 822±96 kg (range 670–1025 kg). For the trials included in the study, the mean (±s.d.) ambient pressure was 101.4±0.5 kPa (range 99.2–102.4 kPa), while the air temperature and humidity (thermometer and hygrometer OH513 Oh Haus & Co.) were 21.3±3.1°C (16–27.6°C) and 74.1±8.3% (57–99%), respectively. The water temperature at the facility housing the animals was 15.9±0.9°C (14.5–20.1°C). All experiments were approved by the Animal Care and Welfare Committee of Fundación Oceanogràfic de la Comunitat Valenciana (animal care number: OCE-19-16) and the US Navy Bureau of Medicine and Surgery (BUMED NRD-910).

Respiratory flow measurements

was measured using a custom-made Fleisch type pneumotachometer (Mellow Design, Valencia, Spain) with a dead space of 700 ml. A soft silicone ring at the base of the pneumotachometer was designed to fit comfortably around the nostrils of the walrus. During data collection, the pneumotachometer was placed over the snout and gently pressed down to prevent leaks around the silicone base. The walrus was trained to close its mouth during data collection to ensure respiration only through the nostrils, and the trainer positioned one hand over the mouth, which allowed any possible leaks to be detected. A low-resistance laminar flow matrix (Z9A887-2, Merriam Process Technologies, Cleveland, OH, USA) was placed inside the pneumotachometer, which created a resistance that increased with . This resistance resulted in a pressure difference across the flow matrix that was measured with a differential pressure transducer (Spirometer Pod, ML 311, ADInstruments, Colorado Springs, CO, USA) connected to the pneumotachometer via two firm-walled, flexible tubes of 310 cm length and 2 mm internal diameter (i.d.) (see details in Fahlman et al., 2015). The pneumotachometer was calibrated for linearity between flow and resistance before and after each trial by pumping different air flow rates, using a 7 l calibration syringe (Series 4900, Hans-Rudolph Inc., Shawnee, KS, USA) as previously detailed (Fahlman et al., 2019b).

Airway and oesophageal pressure measurements

For estimating the dynamic CL, we measured the oesophageal (Poeso) and airway opening pressures (Pao, cmH2O) during spontaneous breaths (Fahlman et al., 2015; Olsen et al., 1969). An oesophageal balloon catheter (47-9005, Cooper Surgical, Trumbull, CT, USA) was manually inserted into the oesophagus for measuring the Poeso, and a sample port was placed above the nostrils of the walrus for measuring the Pao. The catheter was placed at the level of the heart and inflated with 1.0 ml of air. Both sample lines were connected to a differential pressure transducer (MPX-100 mbar type 339/2, Harvard Apparatus, Holliston, MA, USA) through 288 cm length and 2 mm i.d., firm-walled and flexible tubes.

Data acquisition and processing

Measured differential pressures were passed through an amplifier (TAM-A Transducer Amplifier Module, Harvard Apparatus). The data were captured at 400 Hz using a data acquisition system (Powerlab 8/35, ADInstruments) and displayed on a laptop computer running LabChart (v. 8.1, ADInstruments).

Measured inspiratory (insp) and exp were integrated to estimate inspiratory (VT,insp) and expiratory (VT,exp) tidal volume as previously detailed (Fahlman et al., 2019b; Fahlman et al., 2015). All volumes were converted into standard temperature and pressure dry (STPD; Quanjer et al., 1993), where inhaled air was corrected for ambient humidity and temperature, and exhaled air was assumed to be at 37°C and 100% saturated with water vapour. fR was calculated for each trial using the number of complete breaths divided by the measurement period. The dynamic CL was estimated as VT,insp divided by the tidal change in transpulmonary pressure (PL, cmH2O; PL=PaoPoeso) measured at zero flow, following previous procedures (see fig. 1B in Fahlman and Madigan, 2016). The reference pressure for both Pao and Poeso was the ambient atmospheric pressure (Pamb).

Statistical analysis

For the statistical analysis of spontaneous breaths, only periods of normal and complete breaths were considered, and single exhalations or inhalations were removed as in previous studies (Fahlman and Madigan, 2016; Fahlman et al., 2020b). For analysis of active breaths, we only included the 2–4 largest and most similar manoeuvres for each trial.

The difference between expiratory and inspiratory , VT and duration for spontaneous (paired t-test) and active breaths (Wilcoxon signed-rank test) were analysed using SPSS (v.24.0, released 2016, IBM SPSS Statistics for Windows, IBM Corp., Armonk, NY, USA). The relationship between measured dependent variables (, VT and fR) while spontaneously breathing and Mb was analysed using linear mixed-effects models using the lme function in R (v.3.6.1, http://www.R-project.org/). The individual animal was treated as a random effect, which accounted for the correlation between repeated measurements on the same individual (Littell et al., 1998). Homoscedasticity for all models was confirmed by the Bartlett test, and in case of unequal variances the variable was log10-transformed. Best models of remaining variables were chosen by the log-likelihood (LL) ratio test. The effect of the experimental factor (body position) on measured dependent variables (, VT, Ttot and fR) for spontaneous breaths was analysed using a two-way mixed-effects ANOVA (SPSS), where body position was treated as a fixed effect and individual animal as a random effect (Frederick, 1999). In this study P≤0.05 was considered as significant, and data are presented as means±s.d.

RESULTS

For lung function measurements from the three walruses while they were resting and spontaneously breathing in all body positions, the average trial duration was 3.9±1.1 min (range 2.0–7.2 min, n=45) with a total of 763 complete breaths (Table 1). For separate trials while walruses were sitting, a total of 26 active respiratory manoeuvres from 9 trials and two animals were included in the analysis (Table 1), and 21 spontaneous breaths from 3 trials were used to estimate the dynamic CL in one individual. The breathing pattern in the walrus began with an expiration followed by an inspiration and an end-inspiratory pause.

Inspiratory and expiratory phases

For each animal, the mean Ttot, expiratory (Texp) and inspiratory (Tinsp) duration, exp, insp, VT,exp and VT,insp for spontaneous breaths are reported in Table 2, and for active respiratory manoeuvres the same variables are reported in Table 3.

Table 2.

Lung function for spontaneous breaths in adult Pacific walruses

Lung function for spontaneous breaths in adult Pacific walruses
Lung function for spontaneous breaths in adult Pacific walruses
Table 3.

Lung function for active breaths in adult Pacific walruses

Lung function for active breaths in adult Pacific walruses
Lung function for active breaths in adult Pacific walruses

For spontaneous breaths, the average Ttot was 4.6±1.4 s, where Texp was significantly shorter (2.1±0.7 s) than Tinsp (2.5±0.9 s, paired t-test, t=−16.2, d.f.=762, P<0.01). Mean exp was significantly higher (8.5±2.8 l s−1) than insp (6.4±1.9 l s−1, t=25.3, d.f.=762, P<0.01). VT did not differ between expiration (11.5±4.6 l) and inspiration (11.7±4.6 l, t=−1.74, d.f.=762, P>0.05), and we therefore only report VT,exp when referring to VT for spontaneous breaths, unless otherwise specified.

For active respiratory manoeuvres, average Ttot was 3.6±0.9 s, and Texp was significantly shorter (0.9±0.3 s) than Tinsp (2.7±0.8 s, Wilcoxon signed-rank test, T=351, P<0.01), while average exp was significantly higher (35.9±10.0 l s−1) than insp (10.3±0.8 l s−1, T=0, P<0.01). There were no differences between VT,exp (18.8±5.7 l) and VT,insp (19.8±5.4 l, T=194, P>0.5).

Study of body position

For the 45 selected trials to assess lung function from spontaneous breaths, Ttot did not significantly change with the body position of the animals (sitting 5.4±1.3 s, lying 4.9±0.8 s, water 3.9±0.8 s, two-way mixed-effects ANOVA, F2,4=3.92, P>0.1; Fig. 1). The body position of the animals significantly affected (expiration: F2,4=18.60, P<0.01; inspiration: F­2,4=10.42, P<0.05), where both exp and insp were significantly lower when lying down (expiration: 7.1±1.2 l s−1; inspiration: 5.5±1.1 l s−1) as compared with when in water (expiration: 9.9±1.4 l s−1; inspiration: 7.2±1.2 l s−1, Tukey post hoc test, P<0.001 for all; Fig. 1). Mass-specific VT (sVT, ml kg−1) did not significantly differ between the three body positions (sitting 15.7±2.6 ml kg−1, lying down 13.3±2.5 ml kg−1, water 13.8±2.8 ml kg−1, F­2,4=2.7, P>0.1). Similarly, no differences were detected between body positions for fR (sitting 6.2±2.3 breaths min−1, lying down 8.3±1.7 breaths min−1, water 8.4±2.0 breaths min−1, F2,4=6.07, P>0.05).

Fig. 1.

Respiratory flow()and total breath duration of spontaneous breaths from adult Pacific walruses in different body positions. Mean (±s.d., n=15 for each position) expiratory (exp) and inspiratory flow (insp), and total breath duration (Ttot) measured from three voluntarily participating female Pacific walruses (Odobenus rosmarus divergens) while sitting and supported by the front flippers (sitting), lying down in sternal recumbency (lying down) and in water (water).

Fig. 1.

Respiratory flow()and total breath duration of spontaneous breaths from adult Pacific walruses in different body positions. Mean (±s.d., n=15 for each position) expiratory (exp) and inspiratory flow (insp), and total breath duration (Ttot) measured from three voluntarily participating female Pacific walruses (Odobenus rosmarus divergens) while sitting and supported by the front flippers (sitting), lying down in sternal recumbency (lying down) and in water (water).

General lung function and dynamic lung compliance

For all body positions and spontaneous breaths, the average fR for all trials was 7.6±2.2 breaths min−1 (Table 1). The average sVT while spontaneously breathing was 13.9±5.2 ml kg−1 (range 2.2–32.7 ml kg−1), and the highest (19.5 l s−1) and the largest VT (28.8 l, sVT=32.7 ml kg−1) were measured while floating at the water surface (animal ID: 26005389; Table 2). TLCest was calculated based on data from excised lungs of different species of marine mammals, including juvenile walruses (TLCest=0.135Mb0.92, where Mb is in kg and TLCest is in l; Kooyman, 1973), using the Mb measured during the experimental period (Table 1). The average TLCest was 63.2±8.3 l, and for the three body positions and spontaneous breaths, the VT ranged from 3% to 43% of the TLCest. When performing active respiratory manoeuvres while sitting, the maximal (55.4 l s−1) and the largest VT (31.9 l) were measured for walrus 26005390 (Table 3), and the VT reached 50% of TLCest.

For spontaneous breaths, there was a positive correlation between VT,exp and VT,insp and Mb, with a mass-exponent close to unity (Fig. 2, Table 4). During spontaneous breathing, neither exp nor insp correlated with Mb (Table 4). There was a positive correlation between fR and Mb (Table 4).

Fig. 2.

Relationship betweenbody massand measuredtidal volumeduring spontaneous breathing in adult Pacific walruses. Measured body mass (Mb) and tidal volume (VT) from three voluntarily participating female Pacific walruses (O. rosmarus divergens) in three different body positions: sitting supported by the pectoral flippers (black), lying down (dark grey) and floating in water (light grey). The dashed line represents the regression line based on reported results in Table 4 for expiratory tidal volume (VT,exp). The solid line shows the predicted VT using the allometric equation obtained from a number of marine mammal species (VT=0.0372Mb0.97; Fahlman et al., 2020b), while the dotted line represents the estimated VT for terrestrial mammals at rest (VT=7.69Mb1.04; Stahl, 1967).

Fig. 2.

Relationship betweenbody massand measuredtidal volumeduring spontaneous breathing in adult Pacific walruses. Measured body mass (Mb) and tidal volume (VT) from three voluntarily participating female Pacific walruses (O. rosmarus divergens) in three different body positions: sitting supported by the pectoral flippers (black), lying down (dark grey) and floating in water (light grey). The dashed line represents the regression line based on reported results in Table 4 for expiratory tidal volume (VT,exp). The solid line shows the predicted VT using the allometric equation obtained from a number of marine mammal species (VT=0.0372Mb0.97; Fahlman et al., 2020b), while the dotted line represents the estimated VT for terrestrial mammals at rest (VT=7.69Mb1.04; Stahl, 1967).

Table 4.

Statistical results for linear mixed-effects models for voluntary breaths in adult Pacific walruses

Statistical results for linear mixed-effects models for voluntary breaths in adult Pacific walruses
Statistical results for linear mixed-effects models for voluntary breaths in adult Pacific walruses

The average dynamic CL measured in one walrus (animal ID: 26005388) while spontaneously breathing was 1.09±0.23 l cmH2O−1. As CL varies with lung size (Stahl, 1967), the specific CL (sCL, cmH2O−1) was computed by dividing CL by the minimum air volume (MAV, l), which was estimated to be 7% of total lung capacity (TLC, l), based on previous experiments with excised lungs (Fahlman et al., 2011). The Mb of walrus 26005388 at the time of the measurements was 640 kg, resulting in an average dynamic sCL of 0.32±0.07 cmH2O−1.

The range of exp and insp for spontaneous and active breaths combined was 1.7–55.4 l s−1 and 1.9–16.0 l s−1, respectively (Tables 2 and Table 3). When exp and insp were normalized to TLCest (specific respiratory flow: s, s−1), the s for expiration (sexp) ranged from 0.03 to 0.87 s−1 and that for inspiration (sinsp) ranged from 0.03 to 0.25 s−1. The flow–volume relationship for two animals while sitting showed that exp and insp were constant over all lung volumes during both spontaneous and active breaths (Fig. 3).

Fig. 3.

Comparative flow-volume relationship for adult Pacific walrus, bottlenose dolphin and human. Data from one adult female Pacific walrus (O. rosmarus divergens) in the present study (animal ID 26005390, year of birth 2003, 821±38 kg), one adult male bottlenose dolphin (Tursiops truncatus, age 21 years, 197 kg; Fahlman et al., 2015), and one adult man (fig. 4 of Miller et al., 2005; reproduced with permission of the ERS © 2020). (A) Flow–volume relationship for spontaneous and active respiratory manoeuvres from both species of marine mammals, and for one maximal respiratory manoeuvre from the adult human male. (B) For the same data, respiratory flow and VT were normalized by the maximum measured VT for the represented active manoeuvre (walrus 21.8 l, dolphin 18.6 l, human 4.3 l), which was considered as the behavioural vital capacity (VCB). For each representation, the arrow on the right shows the direction of the expiratory phase at the beginning of the respiratory manoeuvres (positive values of respiratory flow or normalized respiratory flow), while the arrow on the left shows the inspiratory phase (negative values). Spontaneous and active breaths were collected through voluntary participation of the animals, where the walrus was sitting and supported by the pectoral flippers and the dolphin was floating in water.

Fig. 3.

Comparative flow-volume relationship for adult Pacific walrus, bottlenose dolphin and human. Data from one adult female Pacific walrus (O. rosmarus divergens) in the present study (animal ID 26005390, year of birth 2003, 821±38 kg), one adult male bottlenose dolphin (Tursiops truncatus, age 21 years, 197 kg; Fahlman et al., 2015), and one adult man (fig. 4 of Miller et al., 2005; reproduced with permission of the ERS © 2020). (A) Flow–volume relationship for spontaneous and active respiratory manoeuvres from both species of marine mammals, and for one maximal respiratory manoeuvre from the adult human male. (B) For the same data, respiratory flow and VT were normalized by the maximum measured VT for the represented active manoeuvre (walrus 21.8 l, dolphin 18.6 l, human 4.3 l), which was considered as the behavioural vital capacity (VCB). For each representation, the arrow on the right shows the direction of the expiratory phase at the beginning of the respiratory manoeuvres (positive values of respiratory flow or normalized respiratory flow), while the arrow on the left shows the inspiratory phase (negative values). Spontaneous and active breaths were collected through voluntary participation of the animals, where the walrus was sitting and supported by the pectoral flippers and the dolphin was floating in water.

DISCUSSION

The respiratory variables collected in the present study were within the previous ranges measured for other marine mammal species and differed from that reported for terrestrial mammals (see a comparative summary in Table 5). The walruses showed a lower fR than that expected for a similarly sized terrestrial mammal. The breathing pattern in the walruses was similar to that reported in other pinnipeds (Kooyman, 1973), and began with an expiration, followed by an inspiration and a respiratory pause. Our results showed that was lower while lying down than when floating in water, while sVT, Ttot and fR remained the same for all body positions. Measured VT was higher than that estimated for a terrestrial mammal of similar size, increased with Mb with a mass-exponent close to unity, and reached 50% of TCLest during active respiratory manoeuvres. Measured dynamic sCL in one walrus was higher than that reported for terrestrial mammals, and was similar to that previously measured in pinnipeds and cetaceans (Fahlman et al., 2017). The peak sexp for active breaths was lower than that reported for cetaceans and California sea lions, but higher than that estimated for Weddell and grey seals. The flow–volume relationship showed that both exp and insp are maintained over the entire lung volume when performing both spontaneous and active respiratory manoeuvres.

Table 5.

Summary of normalized respiratory variables for a number of resting marine mammals

Summary of normalized respiratory variables for a number of resting marine mammals
Summary of normalized respiratory variables for a number of resting marine mammals

Studies on respiratory function and mechanics in marine mammals have used different approaches. Some studies have used anaesthetized or post-mortem animals and excised tissues (Denison and Kooyman, 1973; Denk et al., 2020; Fahlman et al., 2011, 2014; Kooyman and Sinnett, 1979; Leith et al., 1972; Moore et al., 2011), which may not reflect respiratory function in a realistic biological scenario (Fahlman et al., 2017). Other studies have used restrained and/or involuntarily participating animals (Falke et al., 2008; Gallivan, 1981; Irving et al., 1941; Kerem et al., 1975; Kooyman et al., 1971; Olsen et al., 1969; Reed et al., 1994; Scholander and Irving, 1941; Spencer et al., 1967; Wahrenbrock et al., 1974), probably resulting in stress, which may affect the physiology and breathing patterns. As one important aspect when studying physiology is to minimize confounding variables during data collection, studies on trained animals may help minimize stress during voluntary participation (Fahlman et al., 2017). However, one disadvantage when working with trained animals is that voluntary compliance may differ between days. Also, the influence of the trainer may alter the breathing pattern (e.g. anticipatory behaviour could increase respiratory frequency), causing a bias that has to be assessed. For this reason, the subjects should undergo desensitization to the experimental procedures to ensure that data collection is minimally affected. In addition, it is also important to critically evaluate the data and assess whether the measurement has influenced the subject.

General physiological state of the study subjects

In the present study, the walruses participated in a total of 120 trials to ensure desensitization to the lung function procedure. A subset of these trials was analysed and reported where the animals were calm and breathing normally. Measured fR during the experiments in the present study (7.6±2.2 breaths min−1) was higher than that estimated in resting semi-aquatic and fully aquatic mammals (fR=33Mb−0.42; Mortola and Limoges, 2006), but lower than that predicted for terrestrial mammals of the same size (fR=53.5Mb−0.26; Stahl, 1967). These measurements were close to data obtained from the same animals while floating inside a respirometer (5.9±2.3 breaths min−1; A.B.-E. and A.F., unpublished observations), and to those reported in a single walrus under human care (5.9 breaths min−1; Mortola and Seguin, 2009).

The respiratory minute volume (E=fRVT, l min−1) is commonly used as a measure of the volume of air that is moved in and out of the lungs, and provides an alternative method to evaluate departure from normal ventilatory patterns and whether an increase in fR results in hyperventilation (Prakash, 2015). For the three walruses participating in the present study, we found similar results when comparing the average mass-specific respiratory minute volume (sE, l min−1 kg−1) during exhalation with estimated sE calculated from previous reported data in resting trained marine mammals that were additionally monitored through focal observations (Table 5). Thus, the estimated sE suggests that the walruses were not hyperventilating but may have increased fR during some experimental trials (see fR ranges in Table 1), while at the same time reducing VT to retain a normal E and alveolar minute ventilation. This could also explain the wide range of measured VT from spontaneous breaths (3–43% of TLCest) when compared with previous studies in marine mammals (Table 5), and the obtained mass-exponent for fR (Table 4) when compared with previous allometric equations (Fahlman et al., 2020a; Mortola and Limoges, 2006). We suggest that the variability in the reported fR could be a sign of anticipatory behaviour caused by unintentional conditioning, as the animals were positively reinforced for complete breaths during the desensitization period. Despite this, the average and ranges for respiratory variables in the present study agree with those previously reported for marine mammals. Thus, although working with trained animals could result in a bias that should be considered and critically evaluated, the use of animals in managed care allows for data collection in a controlled environment where animal welfare is a priority. In addition, voluntary participation of trained animals provides a manageable opportunity to obtain measurements that may be difficult and/or ethically challenging to collect from wild megafauna.

The effect of body position

Walruses, like other semi-aquatic species, spend part of their time on land and their respiratory function should be adapted to the two different media. When pinnipeds are lying on land, gravity could be affecting lung function as a result of increased pressure on the thoracic cage (Fahlman and Madigan, 2016). Similarly, the hydrostatic pressure of the water column on the chest could also affect lung function when floating at the water surface, as recently suggested for California sea lions (Fahlman et al., 2020b). We therefore tested lung function in water and on land (sitting and lying) to assess changes in lung function in the Pacific walrus. Obtained sVT, Ttot and fR remained the same for the three body positions, whereas measured was higher when the animals were floating in water as compared with lying down. Thus, obtained exp and insp are similar to those previously reported for California sea lions, where it was suggested that the increased hydrostatic pressure on the chest helped to increase , while Ttot decreased to achieve the same VT and alveolar ventilation as on land (Fahlman et al., 2020b). While in the present study Ttot did not change with body position, the higher obtained when floating in water agrees with the previously suggested pressure effect of the water column.

In marine mammals, the expiratory phase is passive and mainly driven by the elastic recoil of the chest, while the inspiratory phase is active (Fahlman et al., 2017), and respiratory function could be limited by gravity and the increased pressure on the chest when lying on land. Indeed, the reported while lying on land in the present study was lower than when floating in water, which could suggest a possible flow limitation on land. However, the hydrostatic pressure could help assist elastic recoil during expiration, allowing for a passive increase in exp while floating in water, as previously suggested (Fahlman et al., 2020b). While the results of the present study do not provide sufficient evidence of a gravitational alleviation while sitting as compared with lying, we do not discard this possible effect. Therefore, further studies on semi-aquatic species would help confirm the respiratory flow–volume limitations related to the body position and media location in these species.

Tidal volume and compliance

Previous studies have showed that marine mammals have more compliant lungs (i.e. higher CL) and a more flexible chest compared with terrestrial mammals (Table 5) (Fahlman et al., 2017; Olsen et al., 1969; Piscitelli et al., 2013). These anatomical features allow these species to exchange much of their TLC in a single breath, and their VC is close to TLC (Fahlman et al., 2011, 2017; Kooyman and Sinnett, 1979; Piscitelli et al., 2010). However, some studies have shown that, even during respiratory efforts following dives or exercise, VT for most breaths is below VC and only around 20–40% of TLCest (see Table 5 in the present study and fig. 4 in Fahlman et al., 2020b). The measured VT for spontaneous breaths in the present study was lower when compared with the estimated VT for a number of marine mammals (Fig. 2), but was between 3% and 43% of TLCest, and reached 50% of TLCest when performing active respiratory manoeuvres. This range for spontaneous VT is similar to that previously measured in resting marine mammals ranging from 20 to 3600 kg (VT=32–43% of TLCest; Fahlman et al., 2017; Kooyman, 1973), and exceeded the 14% of TLC reported for terrestrial mammals (Table 5). In addition, the measured VT for spontaneous breaths was higher than that estimated for terrestrial mammals (Fig. 2), and correlated with Mb with a mass-exponent close to unity (Table 4) as previously reported in otariids (Fahlman and Madigan, 2016; Fahlman et al., 2020b) and cetaceans (Fahlman et al., 2020a). Similarly, the average measured sVT for spontaneous breaths was higher as compared with that of terrestrial mammals, but was lower than that previously reported (Table 5) and estimated from a number of marine mammals (22 ml kg−1; Mortola and Seguin, 2009).

The measured dynamic sCL from one animal in the present study was higher as compared with previous estimations for land mammals, as previously described in their marine counterparts (see Table 5 in the present study, and table 2 in Fahlman et al., 2017, for more species). While chest compliance was not measured in this study, previous results have shown that the chest in pinnipeds does not significantly contribute to the dynamic values (Fahlman et al., 2014). Thus, our results are consistent with previous studies that suggested an increased ventilatory capacity in marine mammals with larger VT and dynamic sCL as compared with terrestrial mammals, and that most breaths while resting or following active respiratory manoeuvres do not reach TLC.

Respiratory flow and flow–volume relationships

In addition to a flexible thorax and compliant lungs, previous studies on the anatomy and mechanical properties of the respiratory system in marine mammals showed that many species have reinforced conducting airways (Bagnoli et al., 2011; Cozzi et al., 2005; Denison and Kooyman, 1973; Fahlman et al., 2017; Kooyman, 1973; Moore et al., 2014; Piscitelli et al., 2013). These anatomical features would allow for alveolar compression during diving and also adequate gas exchange during high exp and short Ttot as compared with terrestrial mammals (Fahlman et al., 2017; Kooyman and Sinnett, 1982; Piscitelli et al., 2010; Stahl, 1967). However, when comparing exp and Ttot in marine mammals, there appears to be considerable variability (Fahlman et al., 2017; Ponganis, 2011), possibly as a result of the large diversity in the respiratory anatomical and mechanical adaptations within this group of mammals (Fahlman et al., 2017; Kooyman, 1973; Moore et al., 2014; Piscitelli et al., 2010, 2013). The peak s can be used to compare the ventilatory exchange capacity among different species, and previous studies have showed that some cetaceans and the California sea lion exceed reported s for humans (Table 5). While measured exp during active manoeuvres in the present study was higher than that reported for humans (Fig. 3), the maximal peak sexp was between the reported values for humans, cetaceans and the California sea lion and those estimated from available data of phocids (Table 5). Further studies providing the opportunity to correlate mechanical properties and respiratory function in these species would help us to understand their respiratory adaptations and the functional consequences on their exchange capacity.

The reported flow–volume relationships for the Pacific walrus in the present study indicated that the flow during active and spontaneous exhalation is maintained over most of the VT, as previously reported for marine mammals (Fig. 3) (Borque-Espinosa et al., 2020; Fahlman et al., 2019b; Fahlman et al., 2015; Fahlman and Madigan, 2016; Kerem et al., 1975; Kooyman and Cornell, 1981; Kooyman et al., 1975; Kooyman and Sinnett, 1979; Matthews, 1977; Olsen et al., 1969). In contrast, the respiratory mechanics of humans show that the peak expiratory flow during maximal exhalations is effort independent, where the peak occurs at high lung volumes and rapidly drops while lung volume decreases (Fig. 3) (Hyatt et al., 1958; Jordanoglou and Pride, 1968). This flow limitation in humans is related to the increasing flow resistance caused by the compression of the flexible distal airways during emptying of the lungs (Hyatt et al., 1958). Thus, considering the stiffer airways described for marine mammals and the reported flow–volume relationships, it is likely that the flow is not limited by the conducting airways and that the expiration during active and spontaneous respiratory manoeuvres appears to be effort dependent in this group of mammals. However, in a recent study that aimed to assess respiratory health in bottlenose dolphins (Tursiops truncatus) similar to spirometry methods in humans (Clausen, 1982; Crapo, 1994), the flow–volume relationships showed flow limitations associated with obstructive respiratory disease (Borque-Espinosa et al., 2020). While additional studies should be conducted to determine whether this also translates to other marine mammals, we expect that respiratory disease would have similar consequences in other species considering the respiratory similarities among this group. Therefore, increased baseline information of normal lung function and flow–volume dynamics in these species would allow evaluation of the mechanical consequences of respiratory disease. This would be beneficial in terms of gaining a better understanding about the respiratory function limitations in marine mammals, and would enhance our effort in the protection of these species through the development of new diagnostic methods.

Conclusions

With the results presented in the current study, we have provided additional data about the respiratory capacity in another marine mammal, the walrus, which will add to collective information to improve our understanding of respiratory physiology in marine mammals. While we collected data on three adult Pacific female walruses, additional information from animals of different age and sex would be relevant to help improve the understanding of respiratory physiology in this species. The results presented here are in agreement with those reported in California sea lions and suggest a flow limitation when lying on land, and that hydrostatic pressure could help increase exp when resting in water versus on land. Further studies would help confirm the respiratory function limitations when lying on land as compared with sitting. Consequently, we propose that lung function studies in other semi-aquatic marine mammals should evaluate respiratory function both on land and in water.

Acknowledgements

The authors would like to thank all the animal care staff at the Oceanogràfic (Valencia, Spain) who showed enthusiasm during the research collaboration, demonstrated great patience and remained positive throughout the study, making possible the completion of this project. We are grateful to Adm+ and Joan Rocabert for construction of the custom-made pneumotachometers used in the current study. Thanks to several animal care and research interns that gently assisted with equipment and procedures when a hand was needed. A special thanks to Carmen Martinez (Universitat de València) for her kind advice with statistics. We are grateful for the suggestions and comments from the referees that helped improve the final manuscript.

Footnotes

Author contributions

Conceptualization: A.B.-E., A.F.; Methodology: A.B.-E., D.F.-F., A.F.; Software: A.F.; Validation: A.B.-E., A.F.; Formal analysis: A.B.-E., A.F.; Investigation: A.B.-E., D.F.-F., A.F.; Resources: A.F.; Data curation: A.B.-E., A.F.; Writing - original draft: A.B.-E.; Writing - review & editing: D.F.-F., R.C.-A., A.F.; Visualization: A.B.-E.; Supervision: R.C.-A., A.F.; Project administration: A.F.; Funding acquisition: A.F.

Funding

Funding for this project was provided by the Office of Naval Research to A.F. (ONR YIP Award no. N000141410563). Fundación Oceanogràfic de la Comunitat Valenciana provided funding for salary and Oceanogràfic provided access to animals and trainer salary support.

Data availability

The data used in this study are freely available from the Open Science Framework: https://osf.io/qd4cy.

References

Bagnoli
,
P.
,
Cozzi
,
B.
,
Zaffora
,
A.
,
Acocella
,
F.
,
Fumero
,
R.
and
Costantino
,
M. L.
(
2011
).
Experimental and computational biomechanical characterization of the tracheo-bronchial tree of the bottlenose dolphin (Tursiops truncatus)
.
J. Biomech.
44
,
1040
-
1045
.
Bertelsen
,
M. F.
,
Acquarone
,
M.
and
Born
,
E. W.
(
2006
).
Resting heart and respiratory rate in wild adult male walruses (Odobenus rosmarus rosmarus)
.
Mar. Mamm. Sci.
22
,
714
-
718
.
Borque-Espinosa
,
A.
,
Burgos
,
F.
,
Dennison
,
S.
,
Laughlin
,
R.
,
Manley
,
M.
,
Capaccioni Azzati
,
R.
and
Fahlman
,
A.
(
2020
).
Pulmonary function testing as a diagnostic tool to assess respiratory health in bottlenose dolphins Tursiops truncatus
.
Dis. Aquat. Org.
138
,
17
-
27
.
Bostrom
,
B. L.
,
Fahlman
,
A.
and
Jones
,
D. R.
(
2008
).
Tracheal compression delays alveolar collapse during deep diving in marine mammals
.
Respir. Physiol. Neurobiol.
161
,
298
-
305
.
Burki
,
N. K.
(
1981
).
Spirometry and other pulmonary function tests
.
J. Fam. Pract.
12
,
119
-
124
.
Clausen
,
J. L.
(
1982
).
Pulmonary Function Testing Guidelines and Controversies
.
New York, NY
:
Academic Press
.
Cozzi
,
B.
,
Bagnoli
,
P.
,
Acocella
,
F.
and
Constantino
,
M. L.
(
2005
).
Structure and biomechanical properties of the trachea of the striped dolphin Stenella coeruleoalba: evidence for evolutionary adaptations to diving
.
Anat. Rec. Part A Discover. Mol. Cell Evol. Biol.
284A
,
500
-
510
.
Crapo
,
R. O.
(
1994
).
Pulmonary-function testing
.
N. Engl. J. Med.
331
,
25
-
30
.
Denison
,
D. M.
and
Kooyman
,
G. L.
(
1973
).
The structure and function of the small airways in pinniped and sea otter lungs
.
Respir. Physiol.
17
,
1
-
10
.
Denk
,
M.
,
Fahlman
,
A.
,
Dennison-Gibby
,
S.
,
Song
,
Z.
and
Moore
,
M.
(
2020
).
Hyperbaric tracheobronchial compression in cetaceans and pinnipeds
.
J. Exp. Biol.
223
,
jeb217885
.
Fahlman
,
A.
and
Madigan
,
J.
(
2016
).
Respiratory function in voluntary participating Patagonia sea lions (Otaria flavescens) in sternal recumbency
.
Front. Physiol.
7
,
1
-
9
.
Fahlman
,
A.
,
Hooker
,
S. K.
,
Olszowka
,
A.
,
Bostrom
,
B. L.
and
Jones
,
D. R.
(
2009
).
Estimating the effect of lung collapse and pulmonary shunt on gas exchange during breath-hold diving: the Scholander and Kooyman legacy
.
Respir. Physiol. Neurobiol.
165
,
28
-
39
.
Fahlman
,
A.
,
Loring
,
S. H.
,
Ferrigno
,
M.
,
Moore
,
C.
,
Early
,
G.
,
Niemeyer
,
M.
,
Lentell
,
B.
,
Wenzel
,
F.
,
Joy
,
R.
and
Moore
,
M. J.
(
2011
).
Static inflation and deflation pressure-volume curves from excised lungs of marine mammals
.
J. Exp. Biol.
214
,
3822
-
3828
.
Fahlman
,
A.
,
Loring
,
S. H.
,
Johnson
,
S. P.
,
Haulena
,
M.
,
Trites
,
A. W.
,
Fravel
,
V. A.
and
Van Bonn
,
W. G.
(
2014
).
Inflation and deflation pressure-volume loops in anesthetized pinnipeds confirms compliant chest and lungs
.
Front. Physiol.
5
,
1
-
7
.
Fahlman
,
A.
,
Loring
,
S. H.
,
Levine
,
G.
,
Rocho-Levine
,
J.
,
Austin
,
T.
and
Brodsky
,
M.
(
2015
).
Lung mechanics and pulmonary function testing in cetaceans
.
J. Exp. Biol.
218
,
2030
-
2038
.
Fahlman
,
A.
,
Moore
,
M. J.
and
Garcia-Parraga
,
D.
(
2017
).
Respiratory function and mechanics in pinnipeds and cetaceans
.
J. Exp. Biol.
220
,
1761
-
1773
.
Fahlman
,
A.
,
Brodsky
,
M.
,
Miedler
,
S.
,
Dennison
,
S.
,
Ivančić
,
M.
,
Levine
,
G.
,
Rocho-Levine
,
J.
,
Manley
,
M.
,
Rocabert
,
J.
and
Borque Espinosa
,
A.
(
2019a
).
Ventilation and gas exchange before and after voluntary static surface breath-holds in clinically healthy bottlenose dolphins, Tursiops truncatus
.
J. Exp. Biol.
222
,
jeb.192211
.
Fahlman
,
A.
,
Epple
,
A.
,
García-Párraga
,
D.
,
Robeck
,
T.
,
Haulena
,
M.
,
Piscitelli-Doshkov
,
M.
and
Brodsky
,
M.
(
2019b
).
Characterizing respiratory capacity in belugas (Delphinapterus leucas)
.
Respir. Physiol. Neurobiol.
260
,
63
-
69
.
Fahlman
,
A.
,
Borque-Espinosa
,
A.
,
Facchin
,
F.
,
Fernandez
,
D. F.
,
Caballero
,
P. M.
,
Haulena
,
M.
and
Rocho-Levine
,
J.
(
2020a
).
Comparative respiratory physiology in cetaceans
.
Front. Physiol.
11
,
1
-
7
.
Fahlman
,
A.
,
Meegan
,
J.
,
Borque-Espinosa
,
A.
and
Jensen
,
E. D.
(
2020b
).
Pulmonary function and resting metabolic rates in California sea lions (Zalophus californianus) on land and in water
.
Aquat. Mamm.
46
,
67
-
79
.
Falke
,
K. J.
,
Busch
,
T.
,
Hoffmann
,
O.
,
Liggins
,
G. C.
,
Liggins
,
J.
,
Mohnhaupt
,
R.
,
Roberts
,
J. D.
Jr.
,
Stanek
,
K.
and
Zapol
,
W. M.
(
2008
).
Breathing pattern, CO2 elimination and the absence of exhaled NO in freely diving Weddell seals
.
Respir. Physiol. Neurobiol.
162
,
85
-
92
.
Frederick
,
B. N.
(
1999
).
Fixed-, Random-, and Mixed-Effects ANOVA Models: A User-Friendly Guide for Increasing the Generalizability of ANOVA Results
.
Institute of Education Sciences
, ERIC Number: ED426098.
Gallivan
,
G. J.
(
1981
).
Ventilation and gas exchange in unrestrained harp seals (Phoca groenlandica)
.
Comp. Biochem. Phy. A
69
,
809
-
813
.
Hyatt
,
R. E.
,
Schilder
,
D. P.
and
Fry
,
D. L.
(
1958
).
Relationship between maximum expiratory flow and degree of lung inflation
.
J. Appl. Physiol.
13
,
331
-
336
.
Irving
,
L.
(
1939
).
Respiration in diving mammals
.
Physiol. Rev.
19
,
112
-
134
.
Irving
,
L.
,
Scholander
,
P. F.
and
Grinnell
,
S. W.
(
1941
).
The respiration of the porpoise, Tursiops truncatus
.
J. Cell. Physiol.
17
,
145
-
168
.
Jordanoglou
,
J.
and
Pride
,
N. B.
(
1968
).
Factors determining maximum inspiratory flow and maximum expiratory flow of the lung
.
Thorax.
23
,
33
-
37
.
Kerem
,
D. H.
,
Kylstra
,
J. A.
and
Saltzman
,
H. A.
(
1975
).
Respiratory flow rates in the sea lion
.
Undersea Biomed. Res.
2
,
20
-
27
.
Kooyman
,
G. L.
(
1973
).
Respiratory adaptations in marine mammals
.
Am. Zool.
13
,
457
-
468
.
Kooyman
,
G. L.
and
Cornell
,
L. H.
(
1981
).
Flow properties of expiration and inspiration in a trained bottle-nosed porpoise
.
Physiol. Zool.
54
,
55
-
61
.
Kooyman
,
G. L.
and
Sinnett
,
E. E.
(
1979
).
Mechanical properties of the harbor porpoise lung, Phocoena phocoena
.
Respir. Physiol.
36
,
287
-
300
.
Kooyman
,
G. L.
and
Sinnett
,
E. E.
(
1982
).
Pulmonary shunts in Harbor seals and sea lions during simulated dives to depth
.
Physiol. Zool.
55
,
105
-
111
.
Kooyman
,
G. L.
,
Kerem
,
D. H.
,
Campbell
,
W. B.
and
Wright
,
J. J.
(
1971
).
Pulmonary function in freely diving Weddell seals, Leptonychotes weddelli
.
Respir. Physiol.
12
,
271
-
282
.
Kooyman
,
G. L.
,
Norris
,
K. S.
and
Gentry
,
R. L.
(
1975
).
Spout of the gray whale: its physical characteristics
.
Science.
190
,
908
-
910
.
Leith
,
D. E.
,
Lowe
,
R.
and
Gillespie
,
J.
(
1972
).
Mechanics of baleen whale lungs
.
Fed. Proc.
31
,
335
.
Littell
,
R. C.
,
Henry
,
P. R.
and
Ammerman
,
C. B.
(
1998
).
Statistical analysis of repeated measures data using SAS procedures
.
J. Anim. Sci.
76
,
1216
-
1231
.
Matthews
,
R. C.
(
1977
).
Pulmonary Mechanics of California sea Lions, Zalophus californianus
, Vol.
MSc
.
San Diego
:
San Diego State University
.
Miller
,
M. R.
,
Hankinson
,
J.
,
Brusasco
,
V.
,
Burgos
,
F.
,
Casaburi
,
R.
,
Coates
,
A.
,
Crapo
,
R.
,
Enright
,
P.
,
van der Grinten
,
C. P. M.
,
Gustafsson
,
P.
et al. 
(
2005
).
Standardisation of spirometry
.
Eur. Respir. J.
26
,
319
-
338
.
Moore
,
M. J.
,
Hammar
,
T.
,
Arruda
,
J.
,
Cramer
,
S.
,
Dennison
,
S.
,
Montie
,
E.
and
Fahlman
,
A.
(
2011
).
Hyperbaric computed tomographic measurement of lung compression in seals and dolphins
.
J. Exp. Biol.
214
,
2390
-
2397
.
Moore
,
C.
,
Moore
,
M. J.
,
Trumble
,
S.
,
Niemeyer
,
M.
,
Lentell
,
B.
,
McLellan
,
W.
,
Costidis
,
A.
and
Fahlman
,
A.
(
2014
).
A comparative analysis of marine mammal tracheas
.
J. Exp. Biol.
217
,
1154
-
1166
.
Mortola
,
J. P.
and
Limoges
,
M.-J.
(
2006
).
Resting breathing frequency in aquatic mammals: a comparative analysis with terrestrial species
.
Respir. Physiol. Neurobiol.
154
,
500
-
514
.
Mortola
,
J. P.
and
Seguin
,
J.
(
2009
).
End-tidal CO2 in some aquatic mammals of large size
.
Zoology
112
,
77
-
85
.
Olsen
,
C. R.
,
Hale
,
F. C.
and
Elsner
,
R.
(
1969
).
Mechanics of ventilation in the pilot whale
.
Respir. Physiol.
7
,
137
-
149
.
Piscitelli
,
M. A.
,
McLellan
,
W. A.
,
Rommel
,
S. A.
,
Blum
,
J. E.
,
Barco
,
S. G.
and
Pabst
,
D. A.
(
2010
).
Lung size and thoracic morphology in shallow- and deep-diving cetaceans
.
J. Morphol.
271
,
654
-
673
.
Piscitelli
,
M. A.
,
Raverty
,
S. A.
,
Lillie
,
M. A.
and
Shadwick
,
R. E.
(
2013
).
A review of cetacean lung morphology and mechanics
.
J. Morphol.
274
,
1425
-
1440
.
Ponganis
,
P. J.
(
2011
).
Diving mammals
.
Compr. Physiol.
1
,
447
-
465
.
Prakash
,
E. S.
(
2015
).
What is the best definition of the term “hyperventilation”
?
Adv. Physiol. Educ.
39
,
137
-
138
.
Quanjer
,
P. H.
,
Tammeling
,
G. J.
,
Cotes
,
J. E.
,
Pedersen
,
O. F.
,
Peslin
,
R.
and
Yernault
,
J.-C.
(
1993
).
Lung volumes and forced ventilatory flows
.
Eur. Respir. J.
6
,
5
-
40
.
Radeos
,
M. S.
and
Camargo
,
C. A.
Jr.
(
2004
).
Predicted peak expiratory flow: differences across formulae in the literature
.
Am. J. Emerg. Med.
22
,
516
-
521
.
Reed
,
J. Z.
,
Chambers
,
C.
,
Fedak
,
M. A.
and
Butler
,
P. J.
(
1994
).
Gas exchange of captive freely diving grey seals (Halichoerus grypus)
.
J. Exp. Biol.
191
,
1
-
18
.
Reed
,
J. Z.
,
Chambers
,
C.
,
Hunter
,
C. J.
,
Lockyer
,
C.
,
Kastelein
,
R.
,
Fedak
,
M. A.
and
Boutilier
,
R. G.
(
2000
).
Gas exchange and heart rate in the harbour porpoise, Phocoena phocoena
.
J. Comp. Physiol. B.
170
,
1
-
10
.
Scholander
,
P. F.
(
1940
).
Experimental investigations on the respiratory function in diving mammals and birds
.
Hvalrådets Skrifter
22
,
1
-
131
.
Scholander
,
P. F.
and
Irving
,
L.
(
1941
).
Experimental investigations on the respiration and diving of the Florida manatee
.
J. Cell. Physiol.
17
,
169
-
191
.
Spencer
,
M. P.
,
Thomas
,
A.
,
Gornall
,
T. A.
and
Poulter
,
T. C.
(
1967
).
Respiratory and cardiac activity of killer whales
.
J. Appl. Physiol.
22
,
974
-
981
.
Stahl
,
W. R.
(
1967
).
Scaling of respiratory variables in mammals
.
J. Appl. Physiol.
22
,
453
-
460
.
Stirling
,
I.
and
Sjare
,
B.
(
1988
).
Preliminary observations on the immobilization of male atlantic walruses (Odobenus rosmarus rosmarus) with Telazol®
.
Mar. Mamm. Sci.
4
,
163
-
168
.
Wahrenbrock
,
E. A.
,
Maruscha
,
G. F.
,
Elsner
,
R.
and
Kenney
,
D. W.
(
1974
).
Respiration and metabolism in 2 baleen whale calves
.
Mar. Fish. Rev.
36
,
3
-
9
.

Competing interests

The authors declare no competing or financial interests.