ABSTRACT

We measured respiratory flow (), breathing frequency (fR), tidal volume (VT), breath duration and end-expired O2 content in bottlenose dolphins (Tursiops truncatus) before and after static surface breath-holds ranging from 34 to 292 s. There was considerable variation in the end-expired O2, VT and fR following a breath-hold. The analysis suggests that the dolphins attempt to minimize recovery following a dive by altering VT and fR to rapidly replenish the O2 stores. For the first breath following a surface breath-hold, the end-expired O2 decreased with dive duration, while VT and fR increased. Throughout the recovery period, end-expired O2 increased while the respiratory effort (VT, fR) decreased. We propose that the dolphins alter respiratory effort following a breath-hold according to the reduction in end-expired O2 levels, allowing almost complete recovery after 1.2 min.

INTRODUCTION

Marine mammals forage underwater but need to return to the surface to replenish O2 stores and to remove the CO2 produced during dives. While holding their breath underwater, they use the O2 in their blood and tissues to fuel aerobic metabolism, which produces CO2. Consequently, the O2 levels decrease and the CO2 levels increase during a dive (Scholander, 1940). Marine mammals have developed several strategies to increase time underwater and, therefore, foraging efficiency. For example, the O2 stores are increased by elevated haematocrit, blood volume, muscle mass and muscle myoglobin levels (Ponganis, 2015). The lungs in most species are not comparatively larger than those in terrestrial species; instead, deep divers seem to have relatively smaller lungs to minimize gas exchange during diving and thereby decompression sickness (Piscitelli et al., 2010; Scholander, 1940).

Diving mammals also seem to increase foraging efficiency by minimizing time at the surface (Boutilier et al., 2001; Carbone and Houston, 1996; Fahlman et al., 2008; Halsey et al., 2003). The length of time required at the surface is constrained by the efficiency of recovery of blood and tissue gas levels. As the diver returns to the surface, cardiac output, which has been reduced during the dive, begins to increase, and the ascent tachycardia that increases perfusion is thought to help prepare the diver to more efficiently restore O2 and remove CO2 (Thompson and Fedak, 1993). It has also been proposed that this pre-surface tachycardia helps remove dissolved N2, which helps to reduce the risk of gas emboli (Fahlman et al., 2007). Empirical data suggest that the animals incur an O2 debt during a dive bout that is not repaid until the recovery period at the end of the dive bout. The dive behaviours observed in pinnipeds (Fahlman et al., 2008; Kooyman et al., 1973) are consistent with optimal foraging models, which indicate that the length of the surface interval is determined by the need to restore O2 stores (Carbone and Houston, 1996; Halsey et al., 2003; Kramer, 1988). While insufficient uptake of O2 would reduce the aerobic dive duration, insufficient removal of CO2 could result in acid–base balance disturbances and lactate build-up. Thus, it has been proposed that that removal of CO2 is the main driving force regulating surface interval duration (Boutilier et al., 2001; Fahlman et al., 2008; Reed et al., 1994, 2000). Recent reports that marine vertebrates may experience gas emboli and suffer decompression sickness (Fernández et al., 2005, 2017; García-Párraga et al., 2014) also suggest that at times accumulation of blood and tissue N2 levels may limit diving and force animals to extend surface duration of the interval (Fahlman et al., 2014; Hooker et al., 2009). Thus, variation in the surface interval duration may have numerous causes, and changes in dive activity and behaviour may alter the duration at the surface. For example, if a species has to dive deeper to find food, this may result in accumulation of CO2 and N2, which may result in longer surface intervals to remove the additional gas taken up or produced. Consequently, understanding the variables that limit the surface interval and how gases are managed and recovered may help provide clues to improve our understanding of cardiorespiratory limitations.

The recovery time at the surface is reduced by increased cardiac output in bottlenose dolphins (Tursiops truncatus) (Miedler et al., 2015). Changes in ventilation following diving, in contrast, are not as obvious or not reported (Kooyman et al., 1971; Kooyman et al., 1973; Reed et al., 1994). Thus, the surface interval is a complex dynamic state that requires sufficient gas exchange to enable efficient foraging and the interaction between ventilation and perfusion will significantly alter gas exchange.

In the current study, we investigated the changes in respiratory effort [respiratory flow (), breathing frequency (fR) and tidal volume (VT)] and gas exchange (expired O2) in the bottlenose dolphin before and after periods of surface breath-hold ranging from 34 to 292 s. Our results indicate a complex relationship between respiratory effort and end-expired O2 content that helps minimize recovery duration following breath-holds. Thus, bottlenose dolphins appear to vary VT, fR and breath duration depending on breath-hold duration to enhance gas exchange and minimize recovery duration. From our results, we estimate that a 196 kg bottlenose dolphin can restore its O2 stores in approximately 1.2 min, following a surface breath-hold of 157 s.

List of abbreviations
     
  • fR

    breathing frequency

  •  
  • iO2

    instantaneous rate of oxygen consumption

  •  
  • PCO2

    carbon dioxide partial pressure (gas) or tension (fluid)

  •  
  • PO2

    oxygen partial pressure (gas) or tension (fluid)

  •  
  • sVT

    mass-specific tidal volume

  •  
  • TLCest

    estimated total lung capacity

  •  
  • respiratory flow

  •  
  • E

    expired minute ventilation

  •  
  • ­exp

    expiratory flow

  •  
  • insp

    inspiratory flow

  •  
  • O2

    rate of oxygen consumption

  •  
  • VA

    alveolar volume

  •  
  • VD

    dead space volume

  •  
  • VT

    tidal volume

  •  
  • VT,exp

    expiratory tidal volume

  •  
  • VT,insp

    inspiratory tidal volume

  •  
  • ΔO2

    difference between inspired and expired O2

MATERIALS AND METHODS

Animals

Eleven adult bottlenose dolphins, Tursiops truncatus (Montagu 1821), of varying age and size (Table 1) were used for all experiments. The dolphins were housed in a facility in either Hawaii (Dolphin Quest – Oahu) or Las Vegas (Siegfried and Roy's Secret Garden). The ID, body mass (Mb, measured within 2 weeks of each experiment), straight length (rostrum to midline fluke notch) and age of the animals are summarized in Table 1. Prior to initiating the study, animals were desensitized to the equipment and trained for novel research-associated behaviours. Therefore, all experimental trials were performed with the dolphins using operant conditioning and participation by the dolphins was voluntary (the animals were not restrained and could refuse to participate or withdraw at any point during the experimental trial). Each experiment (trial) began with one animal remaining stationary in the water, allowing placement of the appropriate equipment (see fig. 1 in Fahlman et al., 2015), breathing spontaneously while continuous measurements were made for between 210 and 400 s (including a 90 s period during which the dolphin was allowed to calm down, ‘pre-breath-hold’). This approach allowed collection of experimental data on lung function in dolphins that were in a relaxed, physiologically normal state.

Table 1.

Morphometric data and resting metabolic rate and lung function parameters from bottlenose dolphins before a surface breath-hold

Morphometric data and resting metabolic rate and lung function parameters from bottlenose dolphins before a surface breath-hold
Morphometric data and resting metabolic rate and lung function parameters from bottlenose dolphins before a surface breath-hold

Next, the dolphins were trained to turn upside-down (ventrum towards the water surface) and keep their blowhole submerged while holding their breath without moving (‘breath-hold’). Breath-holds of pre-determined duration were terminated by a signal from the trainer. Breath-hold durations were based on the history and comfort level of the individual dolphin. No animal was restrained, so they could terminate the breath-hold and/or withdraw from a trial at any time. At the end of the breath-hold, when the dolphin righted itself, the spirometry equipment was immediately placed over the blowhole, allowing measurement of and expired O2 content (see below) during recovery (‘recovery’). For this study, the recovery period lasted 300 s from the first breath after the apnoeic period (please note that in four trials the recovery terminated early, with a minimum recovery period of 181 s, as the dolphin decided to end the session; see Fig. S1 for the layout of a trial).

Respiratory flow

The procedures and equipment were identical to those used in our previous studies (Fahlman et al., 2015, 2018a,c), and the procedure is briefly summarized here. was measured using a custom-made Fleisch-type pneumotachometer (Micah Brodsky, V.M.D. Consulting, Miami, FL, USA; Mellow Design, Valencia, Spain), which housed a low-resistance laminar flow matrix (item no. Z9A887-2, Merriam Process Technologies, Cleveland, OH, USA). A low-resistance diffuser was added to homogenize the flow (Fahlman et al., 2018c), which helped resolve the difference in calibration factors for inspired (insp) and expired (exp) flow (Fahlman et al., 2015). A differential pressure transducer (Spirometer Pod, ML 311, ADInstruments, Colorado Springs, CO, USA) was connected to the pneumotachometer with two firm-walled, flexible tubes (310 cm length×2 mm i.d.). The differential pressure transducer was connected to a data acquisition system (Powerlab 8/35, ADInstruments), and the data were captured at 400 Hz and displayed on a laptop computer running LabChart (v.8.1, ADInstruments). The differential pressure was used to determine and was calibrated using a 7.0 l calibration syringe (Series 4900, Hans-Rudolph Inc., Shawnee, KS, USA). The signal was integrated and the determined as detailed previously (Fahlman et al., 2015).

A normal breath was considered a respiration that began with an exhalation followed by an immediate inspiration. Some breaths were partial, i.e. only exhalation or inhalation, and these were excluded in the analysis specifically looking at the first breath following the breath-hold.

Respiratory gas composition

The concentration of expired O2 was subsampled via a port in the pneumotachometer and passed through a firm-walled, flexible tube (310 cm length×2 mm i.d.) connected to 30 cm length of 1.5 mm i.d. Nafion tubing, fed into a fast-response gas analyser (ML206, Harvard Apparatus, Holliston, MA, USA) at a flow rate of 200 ml min−1. The gas analyser was connected to the data acquisition system and sampled at 400 Hz. The gas analyser was calibrated before and after the experiment using a commercial mixture of 5% O2, 5% CO2 and 90% N2 (product no. 17L-340, GASCO, Oldsmar, FL, USA). Ambient air was used to check the calibration before and after each experimental trial.

We used and expired O2 to estimate the rate of O2 consumption (O2) as previously detailed (Fahlman et al., 2015). To investigate the dynamics of gas exchange, we calculated the instantaneous O2 (iO2) from the volume of O2 taken up since the last breath.

Mean (±s.d.) air temperature and humidity during trials were 28.2±1.9°C (n=80, range: 22.6–33.0°C) and 62.0±7.0% (44–76%) in Hawaii, and 35.6±5.9°C (n=33, range: 21.3–39.0°C) and 17.1±9.5% (10–45%) in Las Vegas. The average water temperature in the lagoon in Hawaii was 26.0±0.6°C (range: 25.6–26.7°C) and in Las Vegas it was 25.0±0.9°C (range: 22.7–26.0°C).

Data and statistical analysis

We removed the first 90 s of data at the beginning of the trial before the breath-hold from the time the pneumotachometer was placed over the blowhole to allow the animal to settle down. Metabolic data are reported as the mean O2 before a breath-hold. The relationship between a dependent variable and experimental covariates was analysed using linear-mixed effects models (lme, R v.3.1.0, 2014, 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). Best models were chosen by the log-likelihood ratio test against nested models. In this study, P≤0.05 was considered as significant and P≤0.1 was considered a trend. Data are presented as the mean±s.d., unless stated otherwise.

As VT, fR, , end-expired O2 and iO2 decayed exponentially throughout the recovery period, we log10-transformed these variables to linearize the relationship with the number of breaths following the breath-hold. This allowed us to determine how Mb and dive duration affected the recovery following a breath-hold.

Lung health assessment

The six dolphins at the Dolphin Quest site underwent thorough clinical and diagnostic assessment to establish general and pulmonary health within 2 weeks of the experiments. This included radiographic (x-ray) and/or ultrasonographic imaging of the thorax with evaluation by one of two board-certified veterinary radiologists with experience in marine mammal diagnostic imaging. Additional testing included complete blood count, serum chemistry, sedimentation rate and sputum cytology. The health of the five dolphins at Siegfried and Roy's Secret Garden – Las Vegas was evaluated from their medical records, which included routine radiographs every 6 months (except for animal M00001). These routine radiographs were evaluated by one of the veterinary radiologists.

RESULTS

Data from a total of 879 spontaneous breaths before and 1017 breaths following voluntary surface breath-holds were collected from 11 adult male bottlenose dolphins (Tables 1 and 2) housed at Dolphin Quest – Oahu in May 2016 or at Siegfried and Roy's Secret Garden – Las Vegas in May 2015. A total of 42 breath-holds were performed with a mean±s.d. breath-hold duration of 157±60 s (Table 2).

Table 2.

Trial details for each dolphin

Trial details for each dolphin
Trial details for each dolphin

and expired gas concentration before breath-hold

End-expired O2 concentration (Table 1), expiratory and inspiratory duration (expiratory: 370±49 ms; inspiratory: 421±58 ms), (exp: 24.9±6.6 l s−1; insp: 15.5±2.6 l s−1) and VT (expiratory: 4.9±1.5 l; inspiratory: 5.0±1.5 l) did not change with breath number (χ2<2.5, d.f.=1, P>0.1 for all). exp was significantly higher than insp (paired t-test, t-value=7.11, d.f.=10, P<0.01), but there was no difference in inspiratory (VT,insp) or expiratory (VT,exp) VT (paired t-test, d.f.=10, t-value=1.20, P>0.1). The duration of the expiratory phase was significantly shorter than that of the inspiratory phase (paired t-test, d.f.=10, t-value=3.4, P<0.01). The average fR and expired minute ventilation (E) before breath-hold were 5.3±1.5 breaths min−1 (range: 2.3–6.8 breaths min−1) and 23.0±8.8 l min−1 (range: 4.1–38.7 l min−1), respectively. All but two short breath-holds (<60 s) began after a normal respiratory cycle of exhalation followed by inspiration.

The mean O2 before breath-hold was 0.925±0.322 l O2 min−1, and the average mass-specific O2 was 4.9±2.3 ml O2 min−1 kg−1.

and expired gas concentration following breath-hold

Following a breath-hold, neither the inspiratory breath duration (χ2=1.9, d.f.=1, P>0.1, 524±400 ms, n=12) nor total breath duration (χ2=2.1, d.f.=1, P>0.1, 866±447 ms) changed with breath number (Nbreaths) or time to recovery (P>0.1), but there was a slight decrease in expiratory duration with each breath (χ2=8.1, d.f.=1, P<0.01, expiratory duration=348−0.6×Nbreaths).

The end-expired O2 concentration (Fig. 1A), instantaneous O2 (Fig. 1B), VT (Fig. 2A), fR (Fig. 2B), E (Fig. 2C) and (Fig. 3) changed during the recovery period. Following a breath-hold, there was considerable variation in the end-expired O2 concentration (Fig. 1A). The first post-breath-hold breath was successfully sampled from a total of 31 breath-holds, and the end-expired O2 decreased significantly with the duration of the breath-hold (one-way ANOVA, F=4.8, P<0.05) from an average of 15.5±4.0% for a breath-hold less than 1 min to 10.4±3.1% for a breath-hold over 3 min (Fig. 1C). The best equation that explained the variation in the end-expired O2 data included duration of the breath-hold, breath number following the breath-hold and increasing E (Table 3). iO2 decreased with breath number and the duration of the breath-hold, and increased with increasing E (Table 3).

Fig. 1.

Expired O2 concentration and rate of O2 consumption following a static surface breath-hold. (A) End-expired O2 and (B) instantaneous rate of oxygen consumption (iO2) versus breath number following a surface breath-hold for all dolphins and breaths used in the study. Data are separated into three different breath-hold durations: less than 60 s (<60 s), more than 60 s but less than 120 s (<120 s), or more than 120 s (>120 s). Solid and dotted black lines are mean and 95% confidence limits (CL), respectively, before the breath-hold (apnoea). Solid red line is a trend line from the model in Table 3. The trend line is for a 196 kg dolphin, following a 157 s breath-hold, and with a mean expired minute ventilation (E) of 23 l min−1. (C) End-expired O2 for the first breath following a breath-hold against dive duration.

Fig. 1.

Expired O2 concentration and rate of O2 consumption following a static surface breath-hold. (A) End-expired O2 and (B) instantaneous rate of oxygen consumption (iO2) versus breath number following a surface breath-hold for all dolphins and breaths used in the study. Data are separated into three different breath-hold durations: less than 60 s (<60 s), more than 60 s but less than 120 s (<120 s), or more than 120 s (>120 s). Solid and dotted black lines are mean and 95% confidence limits (CL), respectively, before the breath-hold (apnoea). Solid red line is a trend line from the model in Table 3. The trend line is for a 196 kg dolphin, following a 157 s breath-hold, and with a mean expired minute ventilation (E) of 23 l min−1. (C) End-expired O2 for the first breath following a breath-hold against dive duration.

Fig. 2.

Tidal volume, breathing frequency and minute ventilation following a static surface breath-hold. (A) Expired tidal volume (VT,exp), (B) breathing frequency (fR) and (C) E versus breath number following a surface breath-hold for all dolphins and breaths used in the study. Recovery data are separated into breath-hold duration: less than 60 s (<60 s), more than 60 s but less than 120 s (<120 s), or more than 120 s (>120 s). Solid and dotted black lines are mean and 95% CL, respectively, before breath-hold. Solid red line is a trend line from the model in Table 3. The trend line is for a 196 kg dolphin, following a 157 s breath-hold.

Fig. 2.

Tidal volume, breathing frequency and minute ventilation following a static surface breath-hold. (A) Expired tidal volume (VT,exp), (B) breathing frequency (fR) and (C) E versus breath number following a surface breath-hold for all dolphins and breaths used in the study. Recovery data are separated into breath-hold duration: less than 60 s (<60 s), more than 60 s but less than 120 s (<120 s), or more than 120 s (>120 s). Solid and dotted black lines are mean and 95% CL, respectively, before breath-hold. Solid red line is a trend line from the model in Table 3. The trend line is for a 196 kg dolphin, following a 157 s breath-hold.

Fig. 3.

Expired or inspired respiratory flow () versus breath number following a breath-hold for all dolphins and breaths used in the study. Solid and dotted black (expiratory) and grey (inspiratory) lines are mean and 95% CL, respectively, before breath-hold. Solid red and orange lines are trend lines for expiratory and inspiratory flow from the model in Table 3. The trend line is for a 196 kg dolphin, following a 157 s breath-hold.

Fig. 3.

Expired or inspired respiratory flow () versus breath number following a breath-hold for all dolphins and breaths used in the study. Solid and dotted black (expiratory) and grey (inspiratory) lines are mean and 95% CL, respectively, before breath-hold. Solid red and orange lines are trend lines for expiratory and inspiratory flow from the model in Table 3. The trend line is for a 196 kg dolphin, following a 157 s breath-hold.

Table 3.

Results from generalized least square analysis of the data from bottlenose dolphins

Results from generalized least square analysis of the data from bottlenose dolphins
Results from generalized least square analysis of the data from bottlenose dolphins

There was large variation in both the expired and inspired VT following breath-hold, but there were no differences in the mean±s.d. VT,insp (5.0±1.5 l) or VT,exp (4.9±1.5 l) (paired t-test, t-value: 1.3, d.f.=10, P>0.1). We therefore only analysed and report the VT,exp. VT,exp increased with the duration of the breath-hold and with Mb, but decreased with breath number following the breath-hold (Table 3). The VT,exp following the breath-hold, expressed as a percentage of the estimated total lung capacity (TLCest) from an equation based on excised lungs from a range of species (Fahlman et al., 2011; Kooyman, 1973) or from an estimate from a diving dolphin (Ridgway et al., 1969), was 30% (range: 4–106%) and 54% (range: 7–184%), respectively. fR increased with the duration of the breath-hold and decreased with breath number (Table 3). The average inspiratory flow (insp: 15.1±2.5 l s−1) was significantly lower than the average expiratory flow (exp: 27.7±4.8 l s−1, paired t-test, t-value: 7.1, d.f.=10, P<0.01). The average exp (31.7±6.8 l s−1, range 3.3–64.5 l s−1, n=11, t-value: 2.61, P<0.05) and insp (20.2±2.8 l s−1, range 0.8–46.6 l s−1, t-value: 4.14, P<0.01) for the first breath following the breath-hold was significantly greater than the average before the breath-hold (Table 1). Both insp and exp decreased with breath number and scaled allometrically with Mb (Table 3). The inspiratory duration did not change with breath number following a breath-hold (χ2=2.02, P>0.1), but both expiratory and total breath duration increased with the duration of the breath-hold and decreased with breath number (Table 3).

Lung health assessment

Thoracic radiographs showed no evidence of clinically active pulmonary or other intrathoracic disease in any dolphin. In two cases (9FL3 and 99L7), a very mild interstitial pattern was identified on radiographs. Another animal (63H4) had two small pulmonary nodules, suggestive of incidental granulomas in this species. The findings were not considered to be clinically relevant and may indicate incidental age-related changes or very mild fibrosis from a previous pulmonary insult.

Three animals had foci of surface lung pathology characterized ultrasonographically as alveolar-interstitial syndrome (63H4, 99L7 and 9FL3). This finding is abnormal but non-specific, and can be seen with lung fibrosis (i.e. from previous scarring), early pneumonia, atelectasis or pulmonary oedema (Lichtenstein et al., 1997). One other dolphin (01L5) had evidence of marginal lymph node enlargement, suspected to be due to a reactive process. The marginal lymph nodes are the primary lymphatic drainage site for the lung parenchyma in dolphins, and can readily be assessed with ultrasound (Cowan and Smith, 1999; Martony et al., 2017).

At the time of this study, there was no significant respiratory disease present that would require medical therapy for any of the dolphins in this study. This assessment statement was based on the judgement of the attending veterinarians at each facility, physical examination, cytological evaluation and thorough assessment of standard diagnostic laboratory parameters, and radiographic and ultrasonographic evaluation.

DISCUSSION

In the current study, we showed that bottlenose dolphins increase , fR and VT following a static surface breath-hold. The during recovery increased more with increasing breath-hold duration, which helped to reduce the number of breaths before the end-expired O2 had returned to levels equivalent to those before the breath-hold. Our results show that a 196 kg dolphin requires approximately 10 breaths to recover from a 159 s breath-hold, or approximately 1.2 min of recovery. Our results provide information about pulmonary function and gas exchange before and after a breath-hold in clinically healthy dolphins, which can be used as a comparison for wild populations.

One advantage of studying physiological function in animals under managed care is that the health of the study participants can be evaluated and monitored on an individual animal basis. In the current study, we confirmed that all animals were considered clinically healthy based on physical examination by experienced marine mammal veterinarians at each facility, and diagnostic imaging interpreted by one of the veterinary radiologists. In addition, studying trained animals under stimulus control that are participating voluntarily allows researchers to obtain baseline values with minimal stress. While some researchers rightly consider results obtained from animals under human care not to be perfectly representative of their free-ranging counterparts, certain information is virtually impossible to obtain from wild populations where controlled studies are extremely challenging to carry out. For that reason, we previously compared lung function values and metabolic rates in bottlenose dolphins under managed care, with both near-shore and pelagic populations, and have shown that results obtained with trained cetaceans do provide useful physiological information (Fahlman et al., 2015, 2018a,c). Thus, the data from the current study provide baseline lung function estimates from healthy dolphins under a situation with minimal stress.

One disadvantage of studying trained animals is that the training itself has the potential to cause a bias, which can be difficult to assess. For example, the large variation in VT following a breath-hold may be the result of bias stemming from a dolphin anticipating additional requests for specific behaviours from the trainer following the breath-hold. At the beginning of the trial, the initial 90 s of the pre-breath-hold period were removed. Following this, there were no systematic changes in end-expired gas concentration or respiratory variables. If the dolphins had been stressed or uncomfortable with the procedure, we would have expected to see systematic changes with time. In addition, the fR was within the range previously reported for bottlenose dolphins (Fahlman et al., 2015, 2018a,c), suggesting that the data reported prior to a breath-hold (Table 1) were normal.

During a surface breath-hold, gas is exchanged between the lungs and the blood; O2 is taken up and CO2 diffuses from the blood into the lungs. Thus, the pulmonary O2 should be lower following the surface breath-hold and then increase as the blood and tissue O2 levels are restored. Having the dolphins perform the breath-hold at the surface allowed us to determine how the pulmonary O2 changes with minimal impact on ambient pressure and the shunt that develops during diving to depth (Kooyman and Sinnett, 1982; Ridgway et al., 1969). Thus, our experimental design allowed us to specifically look at changes in pulmonary gas content due to the breath-hold, and how the dolphins manage respiration during recovery.

The end-expired O2 of the first breath following a breath-hold decreased with dive duration from an average of 9.4% for a breath-hold of up to 70 s to 6.4% when the breath-hold exceeded 180 s (Fig. 1C). The lowest end-expired O2 was 4.2% following a 212 s breath-hold. Following the first breath, the end-expired O2 increased throughout the recovery period, and increased with increasing E (Fig. 1A, Table 3). There was a significant increase in end-expired O2 and a decrease in iO2, VT, fR and E with increasing recovery time after the breath-hold (Figs 13). The end-expired O2 increased with the number of breaths following the breath-hold and increased with increasing E (Table 3, Figs 1 and 3). Thus, the recovery from a breath-hold was enhanced by increased respiratory effort. The VT and end-expired O2 following a dive were similar to those reported in the bottlenose dolphin [mass-specific VT (sVT)=36–43 ml kg−1, end-expired O2=5.4–13.2%; Ridgway et al., 1969], the harbour porpoise (Phocoena phocoena, sVT=35–41 ml kg−1, end-expired O2≈9.5–13.5%; Reed et al., 2000) and the grey seal (Halichoerus grypus, sVT=30–39 ml kg−1, end-expired O2≈8.6–10.1%; Reed et al., 1994). As dive duration increased, more O2 was consumed, which depleted the O2 stores. Following the breath-hold, the dolphins increased fR and VT to help replenish the O2 stores.

There was considerable variation in the end-expired O2 and E following a breath-hold (Figs 1 and 2), and when all post-breath-hold data were analysed, the end-expired O2 increased with dive duration. While this may seem counterintuitive, the longer breath-holds also caused greater VT, fR and E, which may shorten the recovery and counteract the greater decrease in pulmonary O2 during longer dives (Fig. 1C, Table 3). Thus, the animals compensated for the reduced O2 levels by increasing respiratory effort to reduce recovery time. The end-expired O2 returned to normal values within approximately 10 breaths (Fig. 1A), which is similar to the data reported by Ridgway et al. (1969). Similar responses were observed in the freely diving Weddell seal (Leptonychotes weddellii) where both iO2 (Kooyman et al., 1973) and E (Kooyman et al., 1971) changed throughout the recovery period. In the grey seal, in contrast, changes in VT were not observed following breath-holds (Reed et al., 1994). It is possible that the changes in end-expired O2 with breath number are affected by the changes in VT.

During a breath with a VT that is greater than the dead space volume (VD), the measured O2 content initially decreases to a plateau. This plateau reflects alveolar gas content, which, in a healthy lung, indicates arterial gas tension (West, 2012). If VT<VD, the measured O2 content reflects a mixture of gas in the dead space. Consequently, as VT changed with the number of breaths during recovery, it is possible that the temporal changes seen in end-expired O2 are an artefact of these changes. To evaluate this, we estimated the VD for each dolphin by assuming it was similar to the average minimum air volume (MAV=7% of TLCest) (Fahlman et al., 2011; Kooyman and Sinnett, 1979) measured from water displacement of excised lungs from a range of marine mammals, including dolphins. Using these results, we calculated VA=VTVD, where VA is the volume of VT from the alveolar space. Between 0.5% and 2% (depending on which method was used to obtain TLCest) of all breaths during recovery from a breath-hold were negative, representing breaths of lower volume than the VD. However, while VT changed throughout the recovery period, the proportion of breaths below the VD remained constant with either method. Consequently, the changes in end-expired O2 were not dependent on an increasing proportion of breaths not representing alveolar gas content, and we suggest that the end-expired O2 can be used as a reflection of changes in arterial O2 content. Looking at Fig. 1A, we can estimate that a 196 kg dolphin has recovered its end-expired O2 content after 10 breaths following a 157 s breath-hold, i.e. when the red trend line crosses the average end-expired O2 content before the breath-hold. The duration of this recovery will depend on the fR. From Fig. 2B, we can see that fR is about 8 breaths min−1 during the first 10 breaths, so this dolphin would have recovered its arterial O2 content in about 1.2 min.

Reed et al. (1994) showed end-expired O2 and CO2 recovery curves that changed with time in an opposite pattern in seals. For O2, the levels were initially high, then decreased and finally increased again at the end. It was suggested that this could indicate reduced gas exchange at the lung level and/or reduced tissue perfusion (Reed et al., 1994). Either situation would reduce pulmonary gas uptake, and the partial pressures in the lung would remain high for O2 and low for CO2. As blood flow and gas exchange resume during the post-breath-hold surface interval, the venous oxygen (PO2) and CO2 tension (PCO2) would be lower and higher, respectively, causing a drop in pulmonary O2 and an increase in CO2. As O2 is restored and CO2 is removed, the partial pressures again approach normal values. Interestingly, this U-shaped pattern was not reported in the harbour porpoise (Reed et al., 2000), in the bottlenose dolphins in the current study or in the work by Ridgway et al. (1969). A new hypothesis suggesting how cetaceans control pulmonary gas exchange through management of ventilation–perfusion matching was recently published (García-Párraga et al., 2018). Ventilation–perfusion matching would enable active pulmonary shunting, which would allow cetaceans to selectively access pulmonary gases, facilitating exchange of O2 and CO2 with minimal exchange of N2. Thus, the variation in results may reflect differences in the mechanisms by which marine mammals manage gases during diving.

Past measurements have shown that cetaceans are capable of exchanging as much as 90% of their TLCest (Fahlman et al., 2015, 2017a, 2019; Kooyman and Cornell, 1981; Olsen et al., 1969; Ridgway et al., 1969). From these estimates, numerous studies have assumed that the VT is close to TLCest, and during exercise or during recovery from a dive it may be logical to assume that VT is close to TLCest (Dolphin, 1987a; Fahlman et al., 2016; Folkow and Blix, 1992). However, our results suggest that the average VT during recovery from a breath-hold was only between 30% and 54% of TLCest, depending on which value is used to estimate TLCest. The latter value was based on measured VT (5–6 l) following diving in a 138 kg bottlenose dolphin. By assuming that the measured VT represented a diving lung volume of 80% of total lung capacity, TLCest was 6.9 l (Ridgway et al., 1969). Using this TLCest, the VT during recovery ranged from 7% to 184%. The other equation for TLCest was based on work on excised lungs from a range of marine mammal species (TLCest=0.135Mb0.92) (Fahlman et al., 2011; Kooyman, 1973), resulting in a range of VT from 4% to 106% of TLCest. With either method, it appears that during recovery from a surface breath-hold, VT is mostly considerably lower than TLCest. These results are similar to those of our previous study measuring VT during recovery from a bout of exercise in bottlenose dolphins (Fahlman et al., 2016). Our results indicate that the VT,exp during recovery increased with the length of the surface breath-hold (Table 3, Fig. 2A), but most breaths were still considerably lower than TLCest. Based on these results, it appears that bottlenose dolphins have considerable scope to further increase respiratory effort following a breath-hold. While it is difficult to extrapolate this to freely swimming and diving animals, bottlenose dolphins may be capable of much longer (Fahlman et al., 2018b) or more active breath-holds with similar recovery rates. However, this warrants further study if respiratory variables are to be used to assess field metabolic rate.

Past studies have attempted to estimate field metabolic rate from fR in a number of species by assuming static respiratory values (Armstrong and Siegfried, 1991; Blix and Folkow, 1995; Christiansen et al., 2014; Dolphin, 1987a; Dolphin, 1987b; Folkow and Blix, 1992; Williams and Noren, 2009), or by measuring the variation in VT and O2 exchange fraction (ΔO2=inspired O2−expired O2) following exercise (Fahlman et al., 2016). While this method is attractive in its simplicity, considerable variation in VT and ΔO2 yields extensive error (Fahlman et al., 2016, 2017b; Folkow and Blix, 2017). The results from the current study show a large variation in VT, fR and end-expired gas content following breath-holds ranging from 34 to 292 s. Maximal and average dive durations in shallow-diving near-shore dolphins are within this range (Fahlman et al., 2018b; Wells et al., 2013), and this ecotype may therefore show similar variation following diving. In addition, the change in respiratory effort following longer breath-holds to reduce the duration of recovery resulted in a complicated relationship between VT, end-expired gas content and recovery duration. Our results are from static breath-holds and it is possible that the variability in fR and VT in free-ranging dolphins diving to depth and breathing while swimming is reduced. New methods are being developed that may help estimation of fR and VT in free-ranging marine mammals (Cauture et al., 2019; Sumich, 2001; van der Hoop et al., 2014). These may contribute to our understanding of respiratory effort and expired gas content, allowing us to continue to develop more accurate and useful estimates of field metabolic rate from methods based solely on fR.

Both insp and exp increased immediately following a breath-hold, and decreased throughout the recovery period, but were not affected by the duration of the breath-hold (Fig. 3, Table 3). of the first breath following the breath-hold was significantly higher than the average before the breath-hold (Fig. 3). The expiratory breath duration increased positively with breath-hold duration and decreased during recovery. Thus, the increased VT following a surface breath-hold was achieved by increased breath duration and exp, but only by increasing insp. During voluntary breaths, exhalation is passive and mainly driven by the elastic recoil of the chest, whereas the inspiratory flow is active for both voluntary and forced breaths (see fig. 5A in Fahlman et al., 2017a). As the respiratory effort increases, a greater proportion of the expiratory phase becomes active where the respiratory muscles assist in generating high (see fig. 5B,C in Fahlman et al., 2017a). The relative importance of the diaphragm, cranio-cervical, thoracic and lumbo-pelvic muscles to drive inspiration during rest in dolphins is not known (Cotten et al., 2008; Dearolf, 2003). Understanding the mechanism of respiration both at rest and while actively swimming could provide important information for conservation efforts, and this could be an interesting topic for future studies.

There are conflicting reports in the literature about whether marine mammals dive on inhalation or exhalation, but some studies clearly show that cetaceans dive on full or partial inhalation (Ridgway, 1986; Ridgway and Howard, 1979; Scholander, 1940). Our results concur with this, and most breath-holds began following a normal breath, which in many cases had a higher than normal VT,insp. Following a breath-hold, the initial VT was highly variable and occasionally the dolphins began with a small exhalation and inhalation. In these cases, a second breath of larger volume followed shortly after, but these were exceptions to the pattern. During the study trials, when a trainer signalled the beginning of a breath-hold, most dolphins took a deep breath before initiating the breath-hold. While the deep breath before the breath-hold may be a learned behaviour to extend the breath-hold, this implies a certain level of behavioural control and it is likely that free-ranging animals perform a similar inspiration before the breath-hold. Assessment of respiratory effort in free-ranging animals could be made possible by phonospirometry (Sumich, 2001), or by assessing respiratory sinus arrhythmia and relating these changes to VT,insp (Cauture et al., 2019; Fahlman et al., 2017c).

Conclusions

In the current study, we measured respiratory flow and end-expired O2 content in bottlenose dolphins before and after surface breath-holds. Our data show a complex relationship between respiratory effort (VT, fR, E) and end-expired gas content, suggesting that the dolphins maximized effort following long dives in order to minimize recovery duration. While VT increased following a breath-hold, most breaths were considerably lower than TLCest. Thus, dolphins may have considerable scope to increase respiratory effort following longer or more strenuous breath-holds to rapidly replenish O2 stores.

Acknowledgements

A special thanks to all the Marine Mammal Specialists and staff at Dolphin Quest – Oahu, and Siegfried and Roy's Secret Garden – Las Vegas who made this study possible. Thanks also to Angela Casas Quirante for help with the data analysis. We thank the anonymous referees, who provided constructive criticism that helped improve the manuscript. We thank Dolphin Quest and Mirage for providing support for animals and crew, and for access to resources.

Footnotes

Author contributions

Conceptualization: A.F., M.I., J.R.-L.; Methodology: A.F., M.B., S.M., G.L., J.R.-L., M.M.; Validation: A.F., M.B., J.R.; Formal analysis: A.F., S.D., M.I., J.R., A.B.-E.; Investigation: A.F., S.M., M.I., G.L., J.R.-L., M.M.; Resources: A.F., M.B., J.R.; Data curation: A.F., A.B.-E.; Writing - original draft: A.F.; Writing - review & editing: M.B., S.M., S.D., M.I., G.L., J.R.-L., M.M., J.R., A.B.-E.; Supervision: A.F., M.I., J.R.-L., M.M.; Project administration: A.F.; Funding acquisition: A.F.

Funding

Funding for this project was provided by the Office of Naval Research (ONR YIP Award no. N000141410563). Dolphin Quest provided travel and salary support to M.I.

Data availability

The datasets used in this study are freely available at the following link: https://osf.io/wej32. For any questions about the data format, please contact the corresponding author.

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Competing interests

The authors declare no competing or financial interests.

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