Conclusions and future work recommendations

The assumption that respiration rate is an appropriate proxy for the level of metabolic activity lays the foundation of an important part of fundamental research on the energetics of cetaceans, yet results presented here indicate its accuracy is open to question. The findings of the present study support the essential conclusion drawn previously by Sumich (1994) and Kriete (1995): respiration rate alone, ignoring breath-by-breath variation in O exchange, is not

a reliable metric for producing energetic demand estimates in marine mammals. This study confirmed that respiration timing is crucial for deriving energetic estimations for killer whales from breathing observations; respiration timing in combination with the MR during a breath interval demarcated the O2 store at the time of each breath, which in turn defined the O2

uptake per executive breaths.

The fixed EO2 found by Kriete (1995) applied to estimate MR from breathing rates by Williams

and Noren (2009) resulted in a substantial overestimation of energetic requirements for killer whales compared to the estimates derived by the O2 model with an O2 uptake curve. This

observation questions the utility of using fixed EO2 for studying cetacean energetics in general,

and estimations derived using this method should be questioned and handled with care when drawing firm conclusions. Inaccurate energetic estimations potentially have great influence on estimated food requirements by cetaceans and the evaluation of their impact on marine ecosystems as top predators.

Still, it should be stressed that though the presented O2 model including a set O2 uptake curve

is an improvement on deriving energetic estimates compared to using a fixed EO2, the model is

not yet complete and various aspects of this model should be improved. First, the O2 model

should be validated through quantifying kinematics, O2 usage and breath-by-breath EO2 and VT

from captive killer whales tagged with DTAGs. Blow-expirate of individual sequential breaths of tagged whales should be captured (Kriete, 1995) to quantify gas-exchange (VO2 and maximum

EO2) and VT’s per breath throughout breathing bouts, while locomotion effort of captive whales

is quantified through stroking rate and speed (Goldbogen et al., 2006; Gleiss et al., 2011; Simon et al., 2012). The relationship between EO2 and O2 store at the time of each breath is the

key feature of the model; therefore, it is important to take into account aspects that potentially have impact on this curve shape.

During the present and most former studies on in-situ cetacean energetics VT was assumed to

be constant across breaths. Yet, VT within an individual is not necessarily constant and has

been shown to fluctuate (Spencer et al., 1967; Olsen et al., 1969; Wahrenbrock et al., 1974; Gallivan et al., 1986; Kriete, 1995). Results of this study showed that after long apnea the first breath is most important for O2 store replenishment. Besides, it has been speculated that

required (Wilson, 2003; Wilson et al., 2003; Wilson and Quintana, 2004; Baird et al., 2006).

The rate at which O2 is taken up into the body is not constant and relies not only on the O2

partial pressure in the lungs, but also the O2 partial pressure in the blood and muscles (Butler

and Jones, 1997). In deep-diving cetaceans blood is the major O2 store, while in other

cetaceans the three stores are more closely equal (Kooyman, 2002; Ponganis, 2011). VO2

during prolonged periods of apnea in non-deep-diving cetaceans decreased non-linearly, indicating that the key O2 supply will shift from the lungs to the blood and muscle tissues

(Kramer, 1988; Gallivan, 1992; Kriete, 1995; Noren et al., 2012b). Moreover, after the respiratory tract is reloaded by breathing, the blood and muscle stores will be refilled (Kriete, 1995). This reloading of the compartment during different time intervals is expected to have a significant influence on the true O2 uptake curve. Simultaneously, due to lung collapse at

depth, arterial partial pressure of O2 increases swiftly early in a dive, followed by a fast decline

as gas exchange from the lungs is hindered (Fahlman et al., 2009). Also, marine mammals potentially have the option to voluntarily switch between O2 stores using selective ischemia to

muscles (Scholander, 1940). It is acknowledged that the established O2 model should be

extended into a more complete gas-exchange model by comprising the different body tissues and lung collapse (Fahlman et al., 2006). Still, it is not foreseen this aspect would significantly influence the main outcomes of this study.

For this same reason anaerobic metabolism was not considered for this O2 model; estimated

O2 store was allowed to become negative, but lactate built up was not explicitly taken into

account. Blood lactate level increases due to the depletion of muscle O2 stores triggered either

by exceeding the aerobic breath-hold limit or exercise (Kooyman et al., 1980; Williams et al., 1993). Still, for Noren et al. (2012b) concluded that apnea of 13.3 min in an adult male killer whale did not caused a rise in blood lactate levels, it was assumed that lactate increase concerning breath-hold did not influence analyses outcomes since maximum apnea duration observed was 6.1 and 5.3 min for males and females, respectively. Nevertheless, it is hypothesized that anaerobic metabolism is valuable to include in the future O2 model

concerning short-term anaerobic sprints, for instance during hunting. Moreover, the potential of the established model lies in the fact that it is operational to study also the energetics and respiration of other cetacean species, if adjusted where needed. Extension concerning anaerobic metabolism and the dive response features would make it applicable to deep-diving

cetaceans.

Though the influence by CO2 accumulation during apnea on respiration timing has been

incorporated to diving models for aquatic birds, it has not been applied to models for marine mammals (Wilson et al., 2003; Green et al., 2005; Mortola and Seguin, 2009; Houston, 2011). Also, the removal of CO2 through breathing will influence surface interval times, or the number

of consecutive breaths, as much as O2 replenishment (Boutilier et al., 2001; Wilson et al., 2003;

Green et al., 2005). To improve accuracy of energetic estimates using the established O2