Three adult female Pacific walruses born in 2003 and housed at Oceanografic (Valencia, Spain) Aquarium participated in the study. Prior to initiating the data collection, the animals underwent 7 months of desensitization to the respirometry equipment and were trained to perform the different experiments using operant conditioning. Data collection took place between April 2017 and November 2019. No data were collected during the reproductive season (from around February to March in the study subjects), due to low interest in participation of the study subjects. Body mass (Mb, kg) was measured before the first feeding in the morning the same week of the metabolic trial. Animals identified as "fasted" were postabsorptive during the metabolic measurements and had not had a meal for at least 15h prior to initiating the experimental trials, but some food reinforcement was provided during the last minute of most respirometry trials to help reinforce the required experimental behavior. The energy intake (Kcal) of food items used to reinforce behavior at the end of each metabolic trial was calculated using the measured amount of consumed food (kg) and the measured energetic content for each type of food (Kcal kg-1). This energetic content (gross energy) was estimated through bomb calorimetry. For animals that were not fasted, their energy intake (kcal) during the 15 hours prior to the trial are provided.
In-water metabolic measurements were conducted in a 3 m deep seawater pool with a total water volume of 267 m3. Data collection in water was performed using an open flow respirometry system (see Borque-Espinosa et al. 2021a). The animals were trained to surface into a respirometry dome and breath inside the respiratory dome while floating with minimal movement. The walruses remained floating and moved somewhat while inside the respiratory dome. Consequently, the animals were not completely inactive throughout the experimental trials. The O2 and CO2 were measured during independent trials while 1) floating at the water surface (surface metabolic rate [MRS]), and during 2) stationary breath-hold (stationary diving metabolic rate [DMRS]), or 3) active breath-hold (active diving metabolic rate [DMRA]). The MRS was estimated from a minimum of 5 minutes of data collection while the walrus remained calmly floating inside the respiratory dome. For DMRS measurements, the walrus was asked to submerge to a fixed point at the bottom of the pool and to remain stationary while holding its breath for at least 3 minutes. For the DMRA estimation, the animal swam back and forth between two underwater target poles that were positioned approximately ~7 m apart, until reaching ~90 meters of horizontal subsurface swimming. The walruses swam at a depth of ~1.5 m, and all trials were timed to calculate the swimming velocity. To determine either DMRS or DMRA, the walrus was guided to surface inside the respirometry dome at completion of the dives to measure the O2 and CO2 during the post-dive recovery period. The post-dive recovery period ended when the O2 and CO2 remained stable and had returned to similar measured MRS values. Exploratory trials prior to data collection showed that the O2 and CO2 returned to baseline MRS values between 4-5 min and 6-7 min for the stationary and active dives, respectively (see an example in Figure 1 in Borque-Espinosa et al. 2021a). Therefore, to ensure the inclusion of the entire physiological recovery for all the trials, the post-dive recovery period was extended to 5 minutes and 7 minutes for the stationary and active dives, respectively.
The open-flow respirometry system used with the respiratory dome consisted of a vacuum pump (FlowKit Mass Flow Generator, FK-500-1, Sable Systems Int., Las Vegas, NV, USA) pulling air through a floating transparent Plexiglas dome of 120 cm internal diameter (i.d.) via an 800 cm length and 4.5 cm i.d. plastic corrugated tube. The dome was made buoyant by attaching polyethylene foam to the base. A flow-through rate of 500 l min-1 of air assured that the O2 and CO2 were maintained within the dome at greater than or equal to 19% and 2%, respectively. The O2 and CO2 content were measured using a fast-response gas analyzer (Gemini Respiratory Gas Analyzer, part no. 14-1000, CWE Inc., Allentown, PA, USA), which pulled a subsample of the outlet air from the corrugated tube at a flow rate of 200 ml min-1 via a 310 cm length and 2 mm i.d. firm walled, flexible tubing. This flexible tubing was attached to a hydrophobic filter (13 mm i.d.), followed by a 60 cm length and 1.5 mm i.d. Nafion© sample line connected to the gas analyser. Both, respiratory gas concentrations and air flow rates were captured at 400 Hz using a data acquisition system (Powerlab 8/35, ADInstruments, Colorado Springs, CO, USA) and displayed on a laptop computer running Labchart (v. 8.1, ADInstruments). The effective volume of the system was 465 l, which resulted in a time constant of 0.93 min. The time required to reach a 95% fractional transformation to a new steady state was 167 s (2.79 min) or 3 times the time constant. The measured humidity outside and inside the dome were, respectively, 71 ± 7% (54 – 82%) and 69 ± 7% (53-81%), while the ambient pressure and air temperature at the facility housing the animals during in-water trials were 101.8 ± 0.4 kPa (100.9-102.6 kPa) and 19.2 ± 2.1°C (15.4-23.1°C), respectively. The water temperature averaged 15.4 ± 1.1°C. Oxygen consumption (VO2) and carbon dioxide production (V CO2) was measured per unit of time where the time represented the period in which the walrus was resting and measurements were collected or, for active behaviors, the period in which the behavior occurred (i.e., diving or swimming) plus the period in which respiratory measures were made within the dome. The number of full inhalation and exhaltion breaths were counted during the period of measurement for oxygen consumption and carbon dioxide production.
Because we aimed to measure metabolic rates while walruses were also hauled out on land, we could not use a respiratory dome that was used for in-water behaviors. Instead, metabolic rates were measured using breath-by-breath respirometry when walruses were hauled out (Borque-Espinosa et al. 2021b). Experiments began when the walrus remained inactive for at least 5 minutes and took complete breaths for at least 2 minutes and there were no leaks due to movement, as previously detailed (Borque-Espinosa et al. 2021b). Respiratory flow (V̇) was measured using a custom-made Fleisch type pneumotachometer (Mellow Design, Valencia, Spain) that was placed over the snout and pressed down to prevent leaks around a silicon base. The walrus was trained to close its mouth during data collection to ensure respiration occurred only through the nostrils. The trainer positioned one hand over the mouth to allow detection of any leaks. The pneumotachometer was connected to a differential pressure transducer (Spirometer Pod, ML311, ADInstruments, Colorado Springs, CO, USA) with flexible tubing (see Borque-Espinosa et al. 2021b). The pneumotachometer was calibrated for linearity and flow immediately before and after each trial (Borque-Espinosa et al. 2021b). The inspiratory (VTinsp) and expiratory (VTexp) tidal volumes were determined by integrating the expiratory V̇ (V̇exp) and inspiratory (V̇insp) flows, respectively, as previously detailed (Fahlman et al., 2015; Fahlman and Madigan, 2016; Fahlman et al., 2020). To convert all respiratory flows and volumes into standard temperature pressure dry (STPD), the exhaled air was assumed to be at 37ºC and 100% saturated of water vapor while the inhaled air was corrected for the measured ambient temperature, humidity and pressure. The expired and inspired O2 and CO2 compositions were subsampled via a port in the pneumotachometer that was close to the mouth. The gas was passed through a 310 cm length of 2 mm I.D., firm walled, flexible tubing and a 30 cm length of 1.5 mm i.d. Nafion tubing, to a fast-response O2 and CO2 analyzer (Gemini respiratory monitor, CWE Inc) at a flow rate of 200 ml min-1. The respiratory flows and volumes were corrected for flow to the gas analyzer. The gas analyzer was connected to a computer with LabChart data acquisition software (www.adinstruments.com) that sampled gas at 400 Hz. The gas analyzers were calibrated before and after the experiment using a commercial mixture of 5% O2, 5% CO2, and 90% N2, certified accurate to at least 0.01% (Air Products, www.airproducts.com). The expired gas concentrations and V̇exp were phase corrected to account for the delay caused by the flow in the sample line and multiplied to calculate the instantaneous O2 consumption and CO2 production rates, as previously described (Fahlman et al., 2015; Fahlman and Madigan, 2016; Fahlman et al., 2020). The total volume of O2 and CO2 exchanged during each breath was calculated by integrating the instantaneous VCO2 and VO2 during expiration. The overall VO2 and VCO2 for each trial were estimated by adding the volume for all breaths, which was then divided by the trial duration. The number of full inhalation and exhalation breaths were counted and recorded for each trial.