J Comp Physiol B (2014) 184:1065–1076 DOI 10.1007/s00360-014-0854-8 ORIGINAL PAPER Living in the fast lane: rapid development of the locomotor muscle in immature harbor porpoises (Phocoena phocoena) Shawn R. Noren · Dawn P. Noren · Joseph K. Gaydos Received: 10 April 2014 / Revised: 29 July 2014 / Accepted: 5 August 2014 / Published online: 23 August 2014 © Springer-Verlag Berlin Heidelberg (outside the USA) 2014 Abstract Cetaceans (dolphins and whales) are born into the aquatic environment and are immediately challenged by the demands of hypoxia and exercise. This should promote rapid development of the muscle biochemistry that supports diving, but previous research on two odontocete (toothed whales and dolphins) species showed protracted postnatal development for myoglobin content and buffering capacity. A minimum of 1 and 1.5 years were required for Fraser’s (Lagenodelphis hosei) and bottlenose (Tursiops truncatus) dolphins to obtain mature myoglobin contents, respectively; this corresponded to their lengthy 2 and 2.5year calving intervals (a proxy for the dependency period of cetacean calves). To further examine the correlation between the durations for muscle maturation and maternal dependency, we measured myoglobin content and buffering capacity in the main locomotor muscle (longissimus dorsi) of harbor porpoises (Phocoena phocoena), a species with a comparatively short calving interval (1.5 years). We found that at birth, porpoises had 51 and 69 % of adult levels for myoglobin and buffering capacity, respectively, demonstrating greater muscle maturity at birth than that found previously for neonatal bottlenose dolphins (10 and 65 %, respectively). Porpoises achieved adult levels for myoglobin and buffering capacity by 9–10 months and 2–3 years postpartum, respectively. This muscle maturation occurred at an earlier age than that found previously for the dolphin species. These results support the observation that variability in the duration for muscular development is associated with disparate life history patterns across odontocetes, suggesting that the pace of muscle maturation is not solely influenced by exposure to hypoxia and exercise. Though the mechanism that drives this variability remains unknown, nonetheless, these results highlight the importance of documenting the species-specific physiological development that limits diving capabilities and ultimately defines habitat utilization patterns across age classes. Keywords Myoglobin · Acid buffering capacity · Diving capacity · Marine mammal · Cetacean · Odontocete Introduction Communicated by H.V. Carey. S. R. Noren (*) Institute of Marine Science, University of California, Center of Ocean Health, 100 Shaffer Road, Santa Cruz, CA 95060, USA e-mail: snoren@ucsc.edu D. P. Noren Conservation Biology Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. East, Seattle, WA 98112, USA J. K. Gaydos UC Davis Wildlife Health Center-Orcas Island Office, 942 Deer Harbor Road, Eastsound, WA 98245, USA Over evolutionary time, the locomotor muscles of aquatic birds and mammals have become specialized to support routine prolonged apneas while swimming and diving. Among these adaptations are elevated myoglobin contents and enhanced buffering capacities compared to terrestrial counterparts (Castellini and Somero 1981; Noren and Williams 2000; Noren 2004). When blood perfusion to a tissue is decreased, oxygen depletion of that area is retarded by the release of myoglobin-bound oxygen into the tissue (Salathe and Chen 1993). When breath-hold duration is prolonged, glycolytic pathways may become increasingly important (Hochachka and Storey 1975; Kooyman et al. 13 1066 1980), during which high buffering capacity in the muscle can counteract changes in pH associated with lactic acid accumulation from anaerobic metabolism (Castellini and Somero 1981). Although it is well known that aquatic birds and mammals have high myoglobin contents and increased muscle buffering capacities (Castellini and Somero 1981; Noren and Williams 2000; Noren 2004), a period of postnatal development is required before these muscle characteristics are mature. The demands of hypoxia and exercise in the aquatic environment should promote rapid development of the muscle biochemistry that supports diving (Kanatous et al. 2009; Geiseler et al. 2013). Yet contrary to this hypothesis, previous research on two odontocetes showed protracted postnatal development of muscle myoglobin content (Dolar et al. 1999; Noren et al. 2001). Other detailed studies of the ontogeny of myoglobin content have predominately focused on aquatic air-breathing species that experience postpartum development while still primarily on land, such as aquatic birds (Haggblom et al. 1988), penguins (Weber et al. 1974; Ponganis et al. 1999; Noren et al. 2001) and pinnipeds (seals and sea lions; Thorson 1993; Noren et al. 2005; Burns et al. 2005, 2007; Richmond et al. 2006; Fowler et al. 2007; Weise and Costa 2007; Lestyk et al. 2009; Verrier et al. 2011). The increases in myoglobin content that accompanied postnatal development in these aquatic animals were attributed to increased exposure to physical activity, thermal demands, and hypoxia (Noren et al. 2001) because these factors were known to increase myoglobin contents in treadmill-conditioned bar-headed geese (Saunders and Fedde 1991), shivering red-backed voles (Morrison et al. 1966), and dive-conditioned tufted ducks and muskrats (Stephenson et al. 1989; MacArthur 1990). However, closer examination of the results across pinnipeds indicates that the duration of the postnatal development period required to attain mature muscle myoglobin content varies across species and generally reflects interspecific variation in the age at independent foraging. For example, hooded seals (Cystophora cristata) achieve 50 % of adult myoglobin levels within their first month of life (Geiseler et al. 2013). This postnatal development period is truncated compared to the postnatal development periods of gray (Halichoerus grypus; Noren et al., 2005) and northern elephant (Mirounga angustirostris; Thorson, 1993) seals. The rapid development in hooded seals corresponds to a short 4-day nursing interval (Bowen et al. 1985) and 1-month post-weaning fast (Bowen et al. 1987). There are also differences in the postnatal development periods across gray and elephant seals that correlate with disparate dependency periods (nursing period plus post-weaning fast period; Noren et al. 2005). While Noren et al. (2005) suggested that the developmental trajectory of myoglobin 13 J Comp Physiol B (2014) 184:1065–1076 in pinnipeds is tightly correlated with life history patterns, the mechanism behind this variation remained unidentified. The postnatal development of gray seals on Sable Island primarily occurred in the absence of hypoxia and exercise since the pups had minimal physical activity and typically stayed out of the water while nursing and fasting (S.R. Noren pers. observ.). More recently, Burns and Hammill (2008) suggested that limited iron availability during the lactation interval may limit oxygen store development in seal pups. A similar interspecific examination of the ontogeny of muscle myoglobin across cetaceans has not yet been conducted. Less is known about the factors that may influence the postnatal development of muscle buffering capacity in marine mammals. For terrestrial mammals, the attainment of mature muscle buffering capacity appears to be rapid (6 days postpartum for sheep; Griffiths et al. 1994) and exposure to hypoxia increases the buffering capacity of expressed dipeptides or proteins (Gore et al. 2001). To date, the full developmental trajectory of muscle buffering capacity has only been investigated in one species of cetacean (Tursiops truncatus; Noren, 2004) and two pinniped species (Cystophora cristata, Pagophilus groenlandicus; Lestyk et al., 2009). Based on these limited datasets, the maturation of buffering capacity appears to be protracted in marine mammals, occurring after the age of weaning, at least 25 days postpartum in seals (Lestyk et al. 2009) to greater than 1.5 years postpartum in dolphins (Noren 2004). An interspecific examination of the ontogeny of muscle myoglobin and buffering capacity in cetaceans is warranted to determine how the developmental trajectory of muscle biochemistry is shaped in this group. Unlike pinnipeds, cetaceans are born directly into the water, and immediately encounter the demands of hypoxia and exercise, which should promote rapid development of muscle biochemistry. Conversely, cetaceans span a wide diversity of life history strategies, where maternal dependency periods range from 8 to 42 months (for review see Evans 1987). If muscle maturation is linked to life history, then there should be variability in the duration required for muscle maturation across cetaceans that correlates with the time to onset of independent foraging. To elucidate the potential link between postnatal development of muscle biochemistry and life history patterns, we examined the developmental trajectory of muscle biochemistry in harbor porpoise (Phocoena phocoena). The life history patterns of harbor porpoises are distinctly different from those of the dolphins explored to date (Table 1). In comparison with other odontocetes, harbor porpoises mature at an earlier age, reproduce more frequently, and have shorter life expectancies (Read and Hohn 1995). Based on this, harbor porpoises have J Comp Physiol B (2014) 184:1065–1076 1067 Table 1 Life history characteristics of harbor porpoise (Phocoena phocoena), Fraser’s dolphin (Lagenodelphis hosei), and bottlenose dolphin (Tursiops truncatus) Average group size Gestation period (months) Length at birth (cm) Lactation period (month) Calving interval (years) Age at sexual maturity (years) Length at sexual maturity (cm) Maximum Length (cm) Harbor porpoise 2–10 11 82a 8 1–2 Fraser’s dolphin 40–800 12.5b 110b ? 2b Bottlenose dolphin 2–25 12 98–130 19 2–3 M: 4a F: 3a M: 7–10b F: 5–8b M: 12 M: 132a F: 155a M: 220–230b F: 210–220b M: 245–260 M: 155.4a F: 177.7a M: 260b F: 250b M: 270 F: 11 F: 220–235 F: 250 Species listed in order of increasing age at sexual maturity Information provided in this table were extracted from Tables 7.2, 8.5, and 8.6 in Evans (1987) except adata from Gearin et al. (1994), where length at birth was the shortest calf and maximum lengths were the longest male and female measured. Data from Gearin et al. (1994) were preferentially used because these harbor porpoises were from the same population as those analyzed in this study b Data from Amano et al. (1996) because data on these characteristics for Fraser’s dolphins were not provided in Evans (1987) Table 2 Age class delineations for the specimen in this study, based on morphological characteristics, body length, and estimated age Age class Age class characteristics Female lengths (cm) Female estimated age (years) Male lengths (cm) Male estimated age (years) Fetus Neonate Calf Juvenile Inutero Recently born Still nursing Weaned, immature 46 78, 83, 85 95 116, 120, 130 NA 0 to <0.25 0.25 to <1 1 to <3 38.5 77, 81, 81, 85, 87, 88, 89 96, 97, 98, 100, 106 116, 117 NA 0 to <0.25 0.25 to <1 1 to <3 Adult Sexually mature 155, 161, 165 ≥3 136, 148, 150, 156 ≥4 been described as living life “in the fast lane”, representing one end of a continuum of odontocete life histories that span a wide diversity of strategies (Read and Hohn 1995). By quantifying age-specific physiological capacities for diving in harbor porpoises we can gain insight into possible inter-age variation in diet, as ontogenetic changes in dive capacity have been associated with ontogenetic variations in diet in other marine mammals (Bowen et al. 1993; Field et al. 2007; Jeglinski et al. 2012). Understanding the physiological constraints for breath-hold diving, and hence habitat use patterns, can allow for predictions of vulnerability to habitat disturbance (Williams et al. 2011). Methods Specimens and muscle collection Harbor porpoise (Phocoena phocoena) specimens (n = 30) examined in this study were acquired opportunistically from strandings, incidental fishery catches, or observed killings by killer whales (Orcinus orca) through the San Juan County Marine Mammal Stranding Network. All specimens were considered to be in good postmortem condition (postmortem condition code 2; Geraci and Lounsbury 2005). The specimens were divided into five age classes based on morphological observations (Table 2) (e.g. presence of umbilicus and/or fetal folds, evidence of sexual maturity) and body length, which provided gross estimates of age based on age estimates of similarly sized specimens from this population [see Tables 4 and 5 in Gearin et al. (1994)]. The sample size for each age class was dependent upon the availability of specimens. Whole carcasses were sampled immediately, stored chilled and sampled within 48 h, or frozen at −7 °C and sampled within 2 months of freezing. Muscle samples were taken from the midbelly of the longissimus dorsi muscle bundle at a site directly anterior to the dorsal fin and stored at −20 °C until muscle biochemical analyses were performed. The longissimus dorsi was chosen as the sampling site because it is the primary locomotor muscle of cetaceans and is one of two muscles that power the upstroke (Pabst 1993). This is the standard site for quantifying muscle biochemistry of the locomotor muscle in cetaceans (Noren and Williams 2000). 13 1068 Muscle biochemical analyses Myoglobin content ([Mb]), reported in g Mb (100 g wet muscle)−1, was determined using the procedure of Reynafarje (1963). Approximately 0.5 g of thawed muscle were minced in a low ionic strength buffer (40 mM phosphate, pH 6.6) and then sonicated (Sonifier Cell Disrupter Model 450, Branson Ultrasonics Corporations, Danbury, CT, USA) for 2–3 min on ice. Buffer to tissue ratio was 19.25 mL buffer per g wet tissue. The samples were centrifuged at −4 °C and 28,000g for 50 min (Sorvall RC—5C Plus superspeed refrigerated centrifuge, DuPont Instruments). The clear supernatant was extracted and then bubbled at room temperature with pure CO for approximately 8 min. To ensure a complete reduction, 0.02 g of sodium dithionite was added. The absorbance of each sample was read at room temperature at 538 and 568 nm on a spectrophotometer (Shimadzu UV–visible recording spectrophotometer UV-160, Shimadzu Corporation, Kyoto, Japan). All samples were run in triplicate. This technique has been used successfully with samples acquired from stranded cetaceans (Noren and Williams 2000; Noren et al. 2001). Muscle buffering capacity (β) due to non-bicarbonate buffers was determined for each muscle sample using the procedures of Castellini and Somero (1981) described below. Thawed samples (~0.5 g) were minced in 10.0 mL normal saline solution (0.9 % NaCl), and sonicated (Sonifier Cell Disrupter Model 450, Branson Ultrasonics Corporations, Danbury, CT, USA) for 3 min on ice. Samples were then titrated at 37 °C with 0.2 N NaOH. Buffering capacity was measured in slykes (µmoles of base required to raise the pH of 1 g wet muscle mass by one pH unit, over the range of pH 6.0–7.0). When pH of a sample was greater than 6.0 before the titration, the pH was lowered by the dropwise addition of 1.0 M HCl. Changes in pH were measured using an accumet basic pH/mV/°C meter (AB15+, Fisher Scientific) with an accumet liquid-filled, glass body single-junction combination pH Ag/AgCl Electrode (13-620-285, Fisher Scientific) and separate ATC probe (13-620-19, Fisher Scientific). Samples were maintained at 37 °C during titration by immersion of the test flask in a warm water bath. All samples were run in triplicate. This technique has been used successfully with samples acquired from stranded cetaceans (Noren 2004). Modeling breath-hold limits To estimate the maximum dive duration for harbor porpoises using aerobic processes, the calculated aerobic dive limit (cADL) was determined for each age class by dividing the calculated total body oxygen store (sum of blood, muscle, and lung oxygen stores) by estimates of metabolic rate following methods described in Kooyman (1989). 13 J Comp Physiol B (2014) 184:1065–1076 Admittedly, the cADL is based on many assumptions that can impact the accuracy of these estimates; however, cADLs have been shown to accurately predict the experimentally determined aerobic dive limit (ADL; Kooyman 1989; Kooyman and Ponganis 1998) when estimates of body oxygen stores and metabolic rate are reliable (Ponganis et al. 1997). We maximized the reliability of our calculations by utilizing species-specific oxygen store data for hemoglobin and myoglobin content and by considering a range of realistic metabolic rates. Details regarding the assumptions that were made for these calculations are provided below. The calculations for the oxygen storage capacity of the blood in L are as follows:   Arterial O2 = (0.33 × BV × m) Hb × 0.00134 L O2 g Hb−1 (0.95 − 0.20 saturation) (1)   Venous O2 = (0.67 × BV × m) Hb × 0.00134 L O2 g Hb−1 [0.95 saturation − (0.05 × 0.95 saturation)] (2) where 0.33 and 0.67 are the estimated proportions of arterial and venous blood, respectively (Lenfant et al. 1970), blood volume (BV) is 0.143 L blood kg−1 based on data from a closely related species (Phocoenoides dalli; Ridgway and Johnston, 1966), and body mass (m) was estimated as 12, 25, 37, and 50 kg for neonate, calf, juvenile, and adult age classes, respectively [based on data in Gearin et al. (1994)]. Hemoglobin content (Hb) is 193 g L−1 based on measurements from two immature harbor porpoises (Reed et al. 2000); one hemoglobin value was used for all age classes because Koopman et al. (1999) did not detect a difference between hemoglobin levels of immature and mature wild porpoises. The oxygen binding capacity of hemoglobin was assumed to be the general value for mammals (0.00134 L O2 g Hb−1; Gregory et al. 1972) and the proportion of saturation and depletion of arterial and venous oxygen reserves are described in detail in Ponganis (2011). The calculation for the oxygen storage capacity of the muscle in L is as follows:   Muscle O2 = Mb × 0.00134 L O2 g Mb−1 (m × p) (3) where age-specific values for myoglobin (Mb) were determined in the present study and converted to g Mb (kg wet muscle)−1, 0.00134 L O2 g Mb−1 is the oxygen binding capacity of Mb (Kooyman 1989), body mass (m) in kg was estimated as described above, and p is the proportion of muscle mass in the body, where p was assumed to be 0.260, 0.295, and 0.330 for the neonate and calf, juvenile, and adult age classes, respectively, based on allometric J Comp Physiol B (2014) 184:1065–1076 1069 ontogeny data presented for harbor porpoises in McLellan et al. (2002). The calculation for the oxygen storage capacity of the lung in L is as follows: Lung O2 = TLV × 0.75 × 0.15 (4) where total lung volume (TLV) in L was calculated from an allometric regression for marine mammals, where TLV = 0.1 × m0.96 (Kooyman 1989) and body mass (m) in kg was estimated as described above. Diving lung volume was assumed to be 75 % of TLV (based on data from dolphins; Goforth 1986), with 15 % representing the oxygen extracted during the dive (Kooyman 1989). The rate at which the oxygen stores are used is essentially the metabolic rate of the animal. Across air-breathing, diving vertebrates, including three odontocete species (Noren et al. 2012), shallow diving emperor penguins (Aptenodytes forsteri; Ponganis et al., 2010) and freely diving Weddell seals (Leptonychotes weddelli; Castellini et al., 1992; Ponganis et al., 1993) a cADL that assumed an oxygen consumption rate of two times Kleiber basal metabolism (BMR, where BMR in LO2 min−1 is approximately 0.0101 × mass0.75; Kleiber 1975) best approximated experimentally determined ADLs. Nonetheless, an elevated diving metabolism may be the best model for the diving capacity of immature marine mammals. Weddell seals (Leptonychotes weddelli) demonstrate a strong developmental effect in diving metabolism, where experimentally determined ADLs for pups, yearlings, and adults were best approximated by cADLs that assumed a diving metabolism of 4, 2, and 1 times BMR, respectively (for review of these data see Schreer et al. 2001). Therefore, we calculated ADLs assuming a metabolic rate of 2 and 4 times BMR as an upper and lower bounds for the dive capacity estimates for each age class. Statistics The relationships between body length (x) and muscle biochemistry (y; either myoglobin content or buffering capacity) were examined using segmented regression analysis (SEGREG, www.waterlog.info) to identify the breakpoint in each of these relationships. The breakpoint represents the body length at which muscle biochemistry plateaus and the muscle has therefore reached mature levels. The selection of the best breakpoint and function type was based on maximizing the statistical coefficient of explanation, and performing tests of significance across seven types of functions (http://www.waterlog.info/pdf/regtxt.pdf). Plots displaying regression analyses of myoglobin content versus buffering capacity and myoglobin content versus buffering capacity–myoglobin ratio were performed using Sigma Plot (Jandel Scientific 10.0). The muscle myoglobin contents and buffering capacities of the five age classes (fetus, neonate, calf, juvenile, and adult) were also compared by one way analysis of variance in combination with an all pairwise multiple comparison procedure (Holm-Sidak method) with a significance level of p < 0.05 (Sigma Stat 3.5, Jandel Scientific). These analyses allow us to interpret the results in terms of life history stage to compare our results to those from other studies. Values are reported as means ± 1 SD. Results Development of muscle biochemistry There were significant differences in myoglobin content (F4,25 = 27.450, P < 0.001) and buffering capacity (F4,25 = 36.184, P < 0.001) across age classes. The myoglobin contents and buffering capacities of neonatal harbor porpoises were 1.30 ± 0.53 g 100 g wet muscle mass−1 and 53.8 ± 4.9 slykes, respectively. This represented 51 and 69 % of the mean adult levels, which were 2.56 ± 0.31 g 100 g wet muscle mass−1 and 78.5 ± 5.5 slykes. For myoglobin content, only fetal and neonatal age classes were significantly lower than older age classes (P < 0.05), while buffering capacity was significantly lower in all age classes compared to adult (P < 0.05). This suggests that mature levels for myoglobin content develop earlier in life than mature levels for buffering capacity. Indeed, the calf age class had achieved 98 % of adult myoglobin levels (Table 3). Closer examination demonstrates that this muscle biochemistry increased linearly with length as the animals grew and matured until a breakpoint occurred at a plateau, representing mature levels (Fig. 1). For harbor porpoises, mature levels of myoglobin content were attained at a body length of 111.9 cm (Fig. 1a). A limited sample size in Gearin et al. (1994) precluded the derivation of an age at length relationship for harbor porpoises in the Washington state region, but according to published age at length relationships for Atlantic harbor porpoises (Gaskin and Blair 1977), a 111.9 cm long animal is approximately 9–10 months old. However, the body length corresponding to the lower 95 % confidence interval for this breakpoint, suggests that myoglobin content levels could mature as early as 1–3 months postpartum in harbor porpoises. These results suggest that myoglobin contents are mature prior to or at the time weaning (8 months postpartum; Table 1). Mature levels of buffering capacity are attained at a body length of 134.6 cm (Fig. 1b). According to age at length relationships for Atlantic harbor porpoises (Gaskin 13 Species Age class (n) % adult length Mb [g (100 g wet muscle−1)] % adult [Mb] Buffering capacity (slykes) % adult buffering capacity 1070 13 Table 3 Development of muscle myoglobin [Mb] and buffering capacity in Cetacea Suborder Odontoceti Family Phoocoenidae Harbor porpoisea (Phocoena phocoena) F (2) N (10) 28 55 0.26 ± 0.05* 1.30 ± 0.53* 10 51 28.8 ± 11.9* 53.8 ± 4.9* 37 69 C (6) J (5) A (7) 65 78 100 2.50 ± 0.48 2.95 ± 0.2 2.56 ± 0.31 98 115 100 60.5 ± 7.3* 69.9 ± 4.3* 78.5 ± 5.5* 77 89 100 49 72 100 45 100 34 90 100 66 100 48 69 100 66 100 52 63 100 0.27 1.58 2.76 0.70 3.58 0.15 2.93 3.45 3.94 5.78 3.00 ± 1.13 5.15 ± 1.20 7.10 ± 0.16 1.83 ± 0.23 5.49 ± 0.10 3.35 6.88 6.82 10 57 100 20 100 4 85 100 68 100 42 73 100 33 100 49 100 100 44.8 67.4 69.1 53.9 ± 2.6 86.5 ± 4.0 34.8 58.9 63.7 75.4 74.9 – – – – – 73.4 77.3 70.7 65 97 100 62 100 55 93 100 100 100 – – – – – 104 109 100 92.3 105.7 98.8 93 107 100 Family Delphinidae Bottlenose dolphinb, c (Tursiops truncatus) Common dolphinb, c (Delphinus capensis) Pacific white-sided dolphinb, c (Lagenorhynchus obliquidens) Striped dolphinb, c (Stenella coeruleoalba) Fraser’s dolphind (Lagenodelphis hosei) Spinner dolphind (Stenella longirostris) Short finned pilot whalee (Globicephala macrorhynchus) Family Ziphiidae Gervais’ beaked whalee, f (Mesoplodon europaeus) N (1) J (1) A (2) 50 86 100 6.55 7.42 7.41 88 100 100 N (1) – 0.13 – 40.8 – J (1) – 0.22 – 47.5 – Suborder Mysticeti Gray whaleb, g (Eschrichtius robustus) *Significantly different then all other age classes a The present study, b Noren et al. (2001), c Noren (2004), Age class mean ± SD calculated from data published in d Dolar et al. (1999), e Velten (2012), f Velten et al. (2013), and g Castellini and Somero (1981) J Comp Physiol B (2014) 184:1065–1076 N (3) J (4) A (5) N (2) A (3) F (1) J (1) A (2) J (1) A (1) C (2) Y (2) A (6) C (3) A (13) C (1) J (1) A (6) 3.0 2.5 2.0 1.5 1.0 0.5 135 80 120 70 105 60 90 50 75 40 60 30 45 20 30 10 0.0 15 0.5 1.0 1.5 2.0 2.5 3.0 Muscle myoglobin content [g (100 g wet muscle) -1] -0.5 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 Body Length (cm) B 90 Muscle acid buffering capacity (slykes) 90 [slykes (g Mb * 100 g wet muscle-1)-1] Myoglobin [g (100g wet muscle) -1] Muscle acid buffering capacity (slykes) A 3.5 1071 Acid buffering capacity:Myoglobin content J Comp Physiol B (2014) 184:1065–1076 80 70 60 50 40 30 20 10 30 40 50 60 70 80 Fig. 2 Muscle buffering capacity in relation to myoglobin content [Mb] throughout development in harbor porpoises. Each dot represents the mean for an individual specimen that was analyzed in triplicate. Age classes are as follows: fetus (white), neonate (white with dot in center of symbol), calf (light gray), juvenile (dark gray), and adult (black). Males and females are denoted by triangles and circles, respectively. Buffering capacity was significantly correlated with myoglobin content (large symbols; dashed line), where Buffering capacity = 12.80 [Mb] + 35.83 (n = 30, r2 = 0.64, F = 50.40, P < 0.001). However, this relationship was not causal; muscles with low myoglobin levels had higher buffering capacity to myoglobin content ratios than myoglobin-rich muscles. This is evident by the negative relationship for myoglobin content and buffering capacity to myoglobin ratio (small symbols; solid line), where buffering capacity to myoglobin ratio = 121.53−1.31 [Mb] + 22.01 (n = 30, r2 = 0.92, F = 154.35, P < 0.0001) 90 100 110 120 130 140 150 160 170 Body Length (cm) Fig. 1 Muscle myoglobin content (a) and buffering capacity (b) of harbor porpoises throughout development. Each dot represents the mean ± SD for an individual specimen that was analyzed in triplicate. Age classes are as follows: fetus (white), neonate (white with dot in center of symbol), calf (light gray), juvenile (dark gray), and adult (black). Males and females are denoted by triangles and circles, respectively. The solid lines represent the linear increases in muscle myoglobin content and buffering capacity with body length (our index of age), until an asymptote for mature muscle biochemistry was achieved. Black boxes show the upper and lower 95 % confidence intervals for both the x and y data. Myoglobin [Mb] content increased according to [Mb] = 0.04 length−1.83 (n = 18, r2 = 0.64, F = 28.55, P < 0.001) until a body length of 111.9 cm was obtained; mean myoglobin content ± SD = 2.72 ± 0.32 g 100 g wet muscle−1 for porpoises ≥111.9 cm in length. Buffering capacity increased according to Buffering capacity = 0.51 length + 10.29 (n = 23, r2 = 0.80, F = 84.62, P < 0.001) until a body length of 134.6 cm was obtained; mean buffering capacity ± SD = 78.54 ± 5.54 slykes for porpoises ≥134.6 cm in length. Details on the segmented regression analysis are described in the “Methods” and Blair 1977), a 134.6 cm porpoise is approximately 2–3 years old. However, the lower 95 % critical interval for this breakpoint suggests that mature buffering capacity could be attained as early as 13 months postpartum. These results indicate that maturation of buffering capacity occurs after weaning but prior to attaining sexual maturity; this is later in life than when mature myoglobin contents were attained (Tables 1, 3). Influence of muscle myoglobin content on buffering capacity Even though the developmental trajectory of myoglobin content and muscle buffering capacity are different, some still might argue that the postnatal development of myoglobin drives changes in buffering ability throughout maturation because myoglobin contributes to the buffering capacity of muscle. Similar to Castellini and Somero (1981) and Noren (2004), we analyzed the relationships between myoglobin content and buffering capacity as well as the relationship between myoglobin content and the ratio of buffering capacity to myoglobin content. Although muscle myoglobin content was correlated with muscle buffering capacity throughout development (Fig. 2), the relationship between myoglobin content and the ratio of buffering capacity to myoglobin content implies that myoglobin content is not the only influence on the buffering capacity of the muscle (Fig. 2). Muscles with low myoglobin levels had an extremely high ratio of buffering capacity to myoglobin content compared to myoglobin-rich muscle, 13 1072 J Comp Physiol B (2014) 184:1065–1076 4.5 6.0 Body oxygen stores (L) 4.0 5.0 3.5 4.0 3.0 (18, 60, 22) 2.5 3.0 2.0 (18, 60, 22) 2.0 1.0 0.0 1.5 (20, 63, 17) 1.0 (22, 68, 10) Calculated aerobic dive limit (min) 7.0 0.5 0.0 Neonate Calf Juvenile Adult Age class Fig. 3 Total body oxygen stores and calculated aerobic dive limits (cADL) of a 12 kg neonate, 25 kg calf, 37 kg juvenile, and 50 kg adult harbor porpoise. Total body oxygen stores are divided into lung (light gray), blood (black), and muscle (dark gray). The percent contribution of each store to the total oxygen store is denoted in parenthesis in the following order: lung, blood, muscle. The black and white circles represent cADLs that assumed an oxygen consumption rate equivalent to two and four times Kleiber metabolic rate, respectively. See the “Methods” for details about the assumptions for these calculations suggesting that myoglobin is not the predominant buffering agent of the muscle. Total body oxygen stores and calculated aerobic dive limits (cADLs) A result of increasing myoglobin content (Table 3) and increasing proportion of muscle mass (McLellan et al. 2002) with maturity in harbor porpoises is an increase in mass-specific muscle oxygen stores with age. We estimated that muscle oxygen stores increase from approximately 4.53 mL O2 kg−1 in neonates to 8.71 mL O2 kg−1 in calves to 11.66 and 11.32 mL O2 kg−1 in juveniles and adults, respectively. Changes in myoglobin content influenced the relative contribution of the different oxygen storage compartments (lung, blood, and muscle) to total body oxygen stores with age. Although the majority of oxygen is stored in the blood of harbor porpoises, the muscle oxygen storage compartment becomes increasingly important with maturation (Fig. 3). Ultimately, enhanced muscle oxygen storage capacity increases total mass-specific stores with age. We estimated that total mass-specific oxygen stores increase from approximately 46.23 mL O2 kg−1 in neonates to 50.12 mL O2 kg−1 in calves to 52.91 and 52.46 mL O2 kg−1 in juveniles and adults, respectively. A result of increased total muscle oxygen stores combined with increased body size and lowered mass-specific metabolic rate is increased dive capacity. Our estimates of dive capacity, calculated aerobic dive limits, increased with ontogeny (Fig. 3). 13 Discussion An important factor in determining the diving capabilities of marine mammals and aquatic birds is the metabolic support at the level of the working skeletal muscle (Hochachka 1986), which includes elevated levels of myoglobin and enhanced buffering capacities in the locomotor muscle compared to terrestrial counterparts (Castellini and Somero 1981). Recent studies have demonstrated that this muscle biochemistry increases throughout ontogeny in penguins and pinnipeds, while studies on cetaceans have been limited due to the difficulties of retrieving samples from animals that spend their entire lives at sea. Although immature muscle biochemistry has been documented in several species of cetacean at birth (Dolar et al. 1999; Noren et al. 2001; Noren 2004; Velten 2012), the full development of this muscle biochemistry has only been described in two species, Fraser’s (Lagenodelphis hosei; Dolar et al., 1999) and bottlenose (Tursiops truncatus; Noren et al., 2001; Noren 2004) dolphins. Based on this, conclusions regarding the developmental trajectory of muscle biochemistry in cetaceans have been limited. Noren et al. (2001) suggested that although cetaceans are born directly into the ocean, the behavior of cetacean calves may mitigate demands that may otherwise drive the maturation of muscle biochemistry. For example, cetacean neonates typically swim in echelon position (calf in close proximity of its mother’s mid-lateral flank), which lowers the effort required by the calf to move at a given swim speed (Noren et al. 2008). Cetacean calves are also nutritionally dependent on their mothers’ milk for prolonged periods (8–42 months depending on the species; for review see Evans 1987) so that the calves do not need to dive to meet their nutritional needs. The distinctly different swimming styles and diving requirements of cetacean calves, relative to adults, alleviate the demands of physical activity and exposure to hypoxia early in life. However, these mitigation strategies early in life cannot fully explain the protracted development of the muscle biochemistry observed for dolphins (Noren et al. 2001; Noren 2004; Table 3). Behavioral observations suggest that dolphins have ample exposure to hypoxia and exercise within the first 6 months postpartum that should drive the rapid maturation of myoglobin. For example, bottlenose dolphin calves are only observed swimming in echelon 11 % of the time by 2 months postpartum as swimming alone increases in importance (Mann and Smuts 1999). Indeed, by 3 months postpartum, the size-specific swim speed (speed body length−1) and the size-specific ability for vertical displacement of the tail flukes (stroke amplitude body length−1) of bottlenose dolphin calves are equivalent to the performance of adults (Noren et al. 2006). Also at 2 months postpartum the calves begin to practice foraging (Mann J Comp Physiol B (2014) 184:1065–1076 20 Myoglobin maturation age (months) and Smuts 1999) and are successful at capturing fish by 4–6 months (Mann and Sargeant 2003). An alternate hypothesis for delayed maturation of the oxygen stores is limited iron availability. Burns and Hammill (2008) suggested that the lactation strategy of pinnipeds impacts iron availability and hence the developmental trajectory of the body oxygen stores. Although dolphin calves nurse throughout the first year of life, by 12 months postpartum solid food is a regular part of their diet (Cockcroft and Ross 1990; Wells 1991; Peddemors et al. 1992; Miles and Herzing 2003; Archer and Robertson 2004). The presence of solid food in the diet can serve to supplement iron levels. Therefore, limited iron availability is an unlikely explanation for the protracted development of the muscle oxygen store in cetaceans (Table 3). Despite sharing common cues (hypoxia and exercise) that drive the maturation of muscle biochemistry, harbor porpoises demonstrate more rapid postnatal development compared to other cetaceans. With the exception of the deep diving Gervais’ beaked whale (Mesoplodon europaeus; Velten 2012), harbor porpoises are born with comparatively greater proportions of adult muscle myoglobin contents and acid buffering capacity (Table 3). The muscle biochemistry of harbor porpoises increases linearly with body length throughout ontogeny until it plateaus at a body length consistent with the length of animals that are 9–10 months postpartum for myoglobin content and 2–3 years postpartum for buffering capacity (Fig. 1). Based on the lower 95 % confidence intervals for these breakpoints, maturation of muscle myoglobin and buffering capacity could occur as early as 1–3 months and 13 months postpartum, respectively (based on the body length of the specimen). In contrast, mature myoglobin contents for bottlenose dolphins are obtained at 1.5–3.4 years postpartum [based on body lengths of the animals in the muscle biochemistry data set in Noren et al. (2001) and age at length curves from Read et al. (1993)], and Fraser’s dolphins achieve mature myoglobin levels at 1–4 years postpartum [based on body lengths of the animals in the muscle biochemistry data set in Dolar et al. (1999) and age at length curves from Amano et al. (1996)]. The development of muscle buffering capacity in bottlenose dolphins is similarly protracted (Noren 2004). These results demonstrate that the maturation time for muscle biochemistry in the locomotor muscle is truncated in harbor porpoises compared to these two dolphin species (Fig. 4). Variability in the degree of maturation at birth and the duration of maturation of the muscle biochemistry across species of odontocetes may be linked to disparate life history patterns. The life history of porpoises has been described as being “in the fast lane” because compared to larger toothed whales, porpoises have early maturity, high reproductive rates, and short life spans (Read and Hohn 1073 Tt 18 16 14 Lh 12 10 Pp 8 16 18 20 22 24 26 28 30 32 Calving interval (months) Fig. 4 Myoglobin maturation duration in relation to calving interval in odontocetes. The three species with ample specimens across age classes (see Table 3) are denoted by their species and genus initials [Lagenodelphis hosei (Dolar et al., 1999), Tursiops truncatus (Noren et al., 2001), and Phocoena phocoena (present study)]. Average calving interval was used as a proxy for the age of independence (see Table 1 for references). This is a reasonable assumption because most cetaceans wean a calf with the birth of a new offspring. The lower 95 % confidence interval of the breakpoint in the relationship for body length versus myoglobin content (as shown in Fig. 1a) was used because it approximates the shortest body length at which mature myoglobin levels were obtained. The age at this length was determined from published species-specific age at length curves [L.h. (Amano et al. 1996), T.t. (Read et al. 1993), and P.p. Gaskin and Blair 1977)] 1995). Although the duration of gestation is similar for harbor porpoises, bottlenose dolphins, and Fraser’s dolphins, the lactation period for harbor porpoises is less than half that of dolphins (Table 1). The accelerated developmental path of the harbor porpoise continues as they attain sexual maturity in 1/3 to 1/2 of the time required for dolphins (Table 1). The relatively mature muscle biochemistry levels at birth in conjunction with rapid attainment of adult levels in harbor porpoises compared to the other odontocete species (Table 3; Fig. 4) are likely to be correlated with their fast-paced life history strategy. This suggests that the developmental trajectory of muscle biochemistry in odontocetes is not solely influenced by exposure to hypoxia and exercise. Some might argue that the accelerated muscular development for the smaller harbor porpoise (54–65 kg; Evans 1987) compared to the two larger dolphin species may be a scaling phenomenon. This is because under equal conditions, smaller mammals typically maintain higher massspecific metabolic rates than larger ones (Kleiber 1975), and presumably have more rapid biochemical reaction rates in general, including those involved in growth and maturation (i.e. muscle development). However, this does not explain the different developmental trajectories in the 13 1074 equally sized dolphin species, bottlenose (150–275 kg; Evans 1987) and Fraser’s (160–210 kg; Evans 1987). Review of studies on pinnipeds also indicates that body size is not correlated with the duration required for the maturation of myoglobin. For example, within phocid seals, the smaller hooded (230–300 kg; Reeves et al. 1992) and gray (200–350 kg; Reeves et al. 1992) seals obtain mature adult myoglobin levels by 1 year postpartum as does the larger northern elephant seal (600–2,000 kg; Reeves et al. 1992). Myoglobin development data are from Lestyk et al. (2009), Noren et al. (2005), and Thorson (1993), respectively. Likewise, within otariids, the smaller California (Zalophus californianus; 110–390 kg; Reeves et al. 1992) and Australian (Neophoca cinerea; 110–300 kg; Reeves et al. 1992) sea lions only obtain 67 and 59 % of adult myoglobin levels, respectively, by 2 years postpartum compared to the larger Steller sea lion (Eumatopias jubatus; 350–1120 kg; Reeves et al. 1992), which obtains 95 % of adult myoglobin levels by 2 years postpartum. Myoglobin development data are from Weise and Costa (2007), Fowler et al. (2007), and Richmond (2004), respectively. Immature harbor porpoises, like other immature marine mammals, are undoubtedly disadvantaged by the requisite need for postnatal development of muscle biochemistry. Harbor porpoises are born with only 51 % of adult myoglobin levels (Table 3). This combined with an increase in the proportion of muscle mass with age (McLellan et al. 2002) limits the ability to store oxygen in muscle, such that estimates of mass-specific muscle oxygen stores for neonates and calves were only 40 and 77 % of that of adults. The increase in oxygen storage capacity from the neonatal to calf stage markedly increased the cADL, an estimate of dive capacity (Fig. 3). Subsequent increases in cADL were predominately associated with an increase in body size, because larger body size facilitates greater total body oxygen stores and lower mass-specific oxygen consumption rates in cetaceans (Noren and Williams 2000). Comparisons of our cADLs to published dive records can provide additional clues to the diving capacity and physiology of harbor porpoises. Westgate et al. (1995) found that maximum dive durations of seven satellite tagged free-ranging harbor porpoises (estimated mass 29.6–70.0 kg) ranged from 3.3 to 5.35 min. Admittedly, it is unknown whether or not the these animals were diving to their full physiological capacity. Nonetheless, this dive performance was approximated by our cADL that assumed a diving metabolism of 2 times Kleiber basal metabolic rate (4.26–6.91 min for a 12 kg neonate to 50 kg adult) (Fig. 4). Interestingly, these cADLs were similar to cADLs of 5.24–5.95 and 4.59–5.21 min, based on the average resting metabolism of two juvenile male harbor porpoises (247 mL O2 min−1 for 28 kg porpoise; Read et al. 1993) and the minimum cost of transport of two female harbor 13 J Comp Physiol B (2014) 184:1065–1076 porpoises (2.41 J kg−1 m−1 at a swim speed of 1.4 m s-1; Otani et al. 2001), respectively. Meanwhile, the cADLs that assumed a diving metabolism of 4× Kleiber basal metabolic rate (2.13–3.45 min for a 12 kg calf to 50 kg adult) underestimated the maximum dive performance of harbor porpoises. Although cADLs are based on numerous assumptions that can affect their precision (see “Methods”), it seems reasonable to assume that a diving metabolism of two times Kleiber is appropriate for harbor porpoises. Indeed, experimentally determined ADLs of other odontocete species were best approximated by cADLs that assumed a diving metabolism of two times Kleiber (Noren et al. 2012). Conclusion We found differences in the developmental patterns for muscle myoglobin content and buffering capacity across odontocetes. Although all cetaceans at birth immediately experience exposure to external cues, such as hypoxia and exercise, that should drive myoglobin development, the level of muscle maturation at birth and the duration required to attain mature levels after birth differed across species. The duration required to attain mature muscle myoglobin contents and acid buffering capacities corresponded with the duration of maternal dependency, with the muscle of harbor porpoises maturing earlier in life compared to other cetaceans. This suggests that the maturation of this muscle biochemistry is primarily linked to life history patterns and is therefore not solely driven by exposure to hypoxia and exercise. The mechanism behind this remains unknown. Regardless, limited mass-specific oxygen reserves in combination with small body size constrain the diving capacity of immature marine mammals. The low mass-specific oxygen stores in neonates and calves contribute to shorter cADLs, which in turn, could influence the foraging behaviors of immature animals and females accompanied by calves. This may result in partitioning of prey resources between immature animals, lactating females, and other members of the population. Acknowledgments Collection of samples was supported in part by funding from the John H. Prescott Marine Mammal and Rescue Assistance Grant through NOAA Fisheries. Analysis of samples was supported by NOAA Northwest Fisheries Science Center. We thank the volunteers and staff at the San Juan Marine Mammal Stranding Network and the Whale Museum, especially A. Traxler, for providing samples for this study. We also thank G. Ylitalo and L. Rhodes and their staff at the NOAA Northwest Fisheries Science Center for providing laboratory equipment and bench space for sample analysis. We thank D. Somo for assistance with sample analysis and M.L. Dolar for providing raw data from Dolar et al. (1999). Finally, we thank the laboratory group of T.M Williams for providing insightful comments on previous versions of this manuscript. Collection of samples from J Comp Physiol B (2014) 184:1065–1076 stranded harbor porpoises was authorized by the NOAA Northwest Regional Office (now the West Coast Regional Office). All experiments comply with the current laws of the United States of America. References Amano M, Miyazaki N, Yanagisawa F (1996) Life history of Fraser’s dolphin, Lagenodelphis hosei, based on a school captured off the Pacific Coast of Japan. Mar Mamm Sci 12(2):199–214 Archer FI, Robertson KM (2004) Age and length at weaning and development of diet of pantropical spotted dolphions Stenella attenuata from the eastern tropical Pacific. Mar Mamm Sci 20:232–245 Bowen WD, Oftedal OT, Boness BJ (1985) Birth to weaning in 4 days: remarkable growth in the hooded seal, Cystophora cristata. Can J Zool 63:2841–2846 Bowen WD, Boness BJ, Oftedal OT (1987) Mass transfer from mother to pup and subsequent mass loss by the weaned pup in the hooded seal, Cystophora cristata. Can J Zool 65:1–8 Bowen WD, Lawson JW, Beck B (1993) Seasonal and geographic variation in the species composition and size of prey consumed by grey seals (Halichoerus grypus) on the Scotian shelf. Can J Fish Aquat Sci 50:1768–1778 Burns JM, Hammill MO (2008) Does iron availability limit oxygen store development in seal pups? In: Proceedings of the 4th CPB meeting in Africa: MARA 2008 “molecules to migration: the pressures of life”, pp 417–428 Burns JM, Costa DP, Frost K, Harvey JT (2005) Development of the body oxygen stores in harbor seals: effects of age, mass, and body composition. Phys Biochem Zool 78(6):1057–1068 Burns JM, Lestyk KC, Folkow LP, Hammill MO, Blix AS (2007) Size and distribution of oxygen stores in harp and hooded seals from birth to maturity. J Comp Physiol B 177:687–700 Castellini MA, Somero GN (1981) Buffering capacity of vertebrate muscle; correlations with potentials for anaerobic function. J Comp Physiol B 143:191–198 Castellini MA, Kooyman GL, Ponganis PJ (1992) Metabolic rates of freely diving weddell seals: correlations with oxygen stores, swim velocity and diving duration. J Exp Biol 165:181–194 Cockcroft VG, Ross JB (1990) Observations on the early development of a captive bottlenose dolphin calf. In: Leatherwood SJ, Reeves R (eds) The bottlenose dolphin. Academic Press, San Diego, pp 461–478 Dolar ML, Suarez P, Ponganis PJ, Kooyman GL (1999) Myoglobin in pelagic small cetaceans. J Exp Biol 202:227–236 Evans PGH (1987) The natural history of whales and dolphins. Christopher Helm Ltd, Bromley Field IC, Bradshaw CJA, van den Hoff J, Burton HR, Hindel MA (2007) Age-related shifts in the diet composition of southern elephant seals expand overall foraging niche. Mar Biol 150(6):1441–1452 Fowler SL, Costa DP, Arnould JPY, Gales NJ, Burns JM (2007) Ontogeny of oxygen stores and physiological diving capability in Australian sea lions. Funct Ecol 21:922–935 Gaskin DE, Blair BA (1977) Age determination of harbor porpoise, Phocoena phocoena (L.), in the western North Atlantic. Can J Zool 55:18–30 Gearin PJ, Melin SR, Delong RL, Kajimura H, Johnson MA (1994) Harbor porpoise interactions with a Chinook salmon set-net fishery in Washington state. In: Perrin WF, Donovan GP, Barlow J (eds) Gillnets and cetaceans. Report of the International Whaling Commission, special issue 15, pp 427–437 Geiseler SJ, Blix AS, Burns JM, Folkow LP (2013) Rapid postnatal development of myoglobin from large liver iron stores in hooded seals. J Exp Biol 216:1793–1798 1075 Geraci JR, Lounsbury VJ (2005) Marine mammals ashore: a field guide for strandings, 2nd edn. National Aquarium, Baltimore 372 pp Goforth HW (1986) Glycogenetic responses and force production characteristics of a bottlenose dolphin (Tursiops truncatus) while exercising against a force transducer. Ph.D. thesis, University of California, Los Angeles, p 137 Gore CJ, Hahn AG, Aughey RJ, Martin DT, Ashenden MJ, Clark SA, Garnham AP, Roberts AD, Slater GJ, McKenna MJ (2001) Live high: train low increases muscle buffering capacity and submaximal cycling efficiency. Acta Physiol Scand 173:275–286 Gregory IC, Hulands GH, Millar RA (1972) On the use of a value for haemoglobin oxygen combining capacity in the indirect determination of blood oxygen content in man. Br J Anaesth 44:222 Griffiths RI, Baldwin J, Berger PJ (1994) Metabolic development of the sheep diaphragm during fetal and newborn life. Respir Phys 95:337–347 Haggblom L, Terwilliger RC, Terwilliger NB (1988) Changes in myoglobin and lactate dehydrogenase in muscle tissues of a diving bird, the pigeon guillemot, during maturation. Comp Biochem Physiol B 91(2):273–277 Hochachka PW (1986) Balancing conflicting metabolic demands of exercise and diving. Fed Proc 45:2948–2952 Hochachka PW, Storey KB (1975) Metabolic consequences of diving in animals and man. Science 187:613–621 Jeglinski JWE, Werner C, Robinson PW, Costa DP, Trillmich F (2012) Age, body mass and environmental variation shape the foraging ontogeny of Galapagos sea lions. Mar Ecol Prog Ser 453:279–296 Kanatous SB, Mammen PPA, Rosenberg PB, Martin CM, White MD, DiMaio JM, Huang G, Muallem S, Garry DJ (2009) Hypoxia reprograms calcium signaling and regulates myoglobin expression. Am J Physiol Cell Physiol 296:C393–C402 Kleiber M (1975) The fire of life: an introduction to animal energetics. Robert E. Krieger Publishing Co., New York Koopman HN, Westgate AJ, Read AJ (1999) Hematology values of wild harbor porpoises (Phocoena phocoena) from the Bay of Fundy, Canada. Mar Mamm Sci 15(1):52–64 Kooyman GL (1989) Diverse divers: physiology and behaviour. Springer, Berlin Kooyman GL, Ponganis PJ (1998) The physiological basis of diving to depth: birds and mammal. Ann Rev Physiol 60:19–32 Kooyman GL, Wahrenbrock EA, Castellini MA, Davis RW, Sinnett EE (1980) Aerobic and anaerobic metabolism during voluntary diving in Weddell seals: Evidence of preferred pathways from blood chemistry and behavior. J Comp Physiol B 138:335–346 Lenfant C, Johanson K, Torrance JD (1970) Gas transport and oxygen storage capacity in some pinnipeds and the sea otter. Respir Physiol 9:277–286 Lestyk KC, Folkow LP, Blix AS, Hammill MO, Burns JM (2009) Development of myoglobin concentrations and acid buffering capacity in harp (Pagophilus groenlandicus) and hooded (Cystophora cristata) seals from birth to maturity. J Comp Physiol B 179:985–996 MacArthur RA (1990) Seasonal changes in the oxygen storage capacity and aerobic dive limits of the muskrat (Ondatra zibethicus). J Comp Physiol B 160:593–599 Mann J, Sargeant B (2003) Like mother, like calf: the ontogeny of foraging traditions in wild Indian Ocean bottlenose dolphins (Tursiops sp.). In: Fragaszy DM, Perry S (eds) The biology of traditions. Cambridge University Press, Cambridge, pp 236–266 Mann J, Smuts B (1999) Behavioral development in wild bottlenose dolphin newborns (Tursiops sp.). Behaviour 136:529–566 McLellan WA, Koopman HN, Rommel SA, Read AJ, Potter CW, Nicolas JR, Westgate AJ, Pabst DA (2002) Ontogenetic allometry and body composition of harbour porpoises (Phocoena phocoena, L.) from the western North Atlantic. J Zool Lond 257:457–471 13 1076 Miles JA, Herzing DL (2003) Underwater analysis of the behavioral development of Atlantic spotted dolphin (Stenella attenuata) calves (birth to 4 years of age). Aquat Mamm 29:363–377 Morrison P, Rosenmann M, Sealander JA (1966) Seasonal variation of myoglobin in the northern red-backed vole. Am J Physiol 211(6):1305–1308 Noren SR (2004) Muscle buffering capacities in cetaceans: Influences of diving performance, swimming performance, body size, and postpartum development. Mar Mamm Sci 20(4):808–822 Noren SR, Williams TM (2000) Body size and skeletal muscle myoglobin of cetaceans: Adaptations for maximizing dive duration. Comp Biochem Physiol A 126:181–191 Noren SR, Williams TM, Pabst DA, McLellan B, Dearolf J (2001) The development of diving in marine endotherms: preparing the skeletal muscles of dolphins, penguins, and seals for activity during submergence. J Comp Physiol B 171:127–134 Noren SR, Iverson SJ, Boness DJ (2005) Development of the blood and muscle oxygen stores in grey seals (Halichoerus grypus): implications for juvenile diving capacity and the necessity of a terrestrial postweaning fast. Physiol Biochem Zool 78(4):482–490 Noren SR, Biedenbach G, Edwards EF (2006) The ontogeny of swim performance and mechanics in bottlenose dolphins (Tursiops truncatus). J Exp Biol 209(23):4724–4731 Noren SR, Biedenbach G, Redfern JV, Edwards EF (2008) Hitching a ride: the formation locomotion strategy of dolphin calves. Funct Ecol 22:278–283 Noren SR, Williams TM, Ramirez K, Boehm J, Glenn M, Cornell L (2012) Changes in partial pressures of respiratory gases during submerged voluntary breath-hold across Odontocetes: Is body mass important? J Comp Physiol B 182(2):299–309 Otani S, Naito Y, Kato A, Kawamura A (2001) Oxygen consumption and swim speed of the harbor porpoise Phocoena phocoena. Fish Sci 67:894–898 Pabst DA (1993) Intramuscular morphology and tendon geometry of the epaxial swimming muscles of dolphins. J Zool Lond 230:159–176 Peddemors VM, Fothergill AW, Cockcroft VG (1992) Feeding and growth in a captive born bottlenose dolphin, Tursiops truncatus S. Afr J Zool 37:74–80 Ponganis PJ (2011) Diving mammals. Compr Phys 1:517–535 Ponganis PJ, Kooyman GL, Castellini MA (1993) Determinants of the aerobic dive limit of Weddell seals: analysis of diving metabolic rates, postdive end tidal PO2’s and blood and muscle oxygen stores. Phys Zool 66:732–749 Ponganis PJ, Kooyman GL, Baranov EA, Thorson PH, Steward BS (1997) The aerobic submersion limit of Baikal seals, Phoca sibirica. Can J Zool 75:1323–1327 Ponganis PJ, Starke LN, Horning M, Kooyman GL (1999) Development of diving capacity in emperor penguins. J Exp Biol 202:781–786 Ponganis PJ, Meir JU, Williams CL (2010) Oxygen store depletion and the aerobic dive limit in emperor penguins. Aquat Biol 8:237–245 Read AJ, Hohn AA (1995) Life in the fast land: the life history of harbor porpoises from the Gulf of Maine. Mar Mamm Sci 11(4):423–440 13 J Comp Physiol B (2014) 184:1065–1076 Read AJ, Wells RS, Hohn AA, Scott MD (1993) Patterns of growth in wild bottlenose dolphins, Turisops truncatus. J Zool Lond 231:107–123 Reed JZ, Chambers C, Hunter CJ, Lockyer C, Kastelein R, Fedak MA, Boutilier RG (2000) Gas exchange and heart rate in the harbour porpoise Phocoena phocoena. J Com???p Physiol B 170:1–10 Reeves RR, Stewart BS, Leatherwood S (1992) The Sierra Handbook of Seals and Sirenians. Sierra Club Books, San Francisco Reynafarje B (1963) Simplified method for the determination of myoglobin. J Lab Clin Med 61(1):138–145 Richmond JP (2004) Ontogeny of total body oxygen stores and aerobic dive potential in the steller sea lion (Eumetopias jubatus). Masters Thesis, University of Alaska, Anchorage, p 114 Richmond JP, Burns JM, Rea LD (2006) Ontogeny of total body oxygen stores and aerobic dive potential in Steller sea lions (Eumetopias jubatus). J Comp Physiol B 176:535–545 Ridgway SH, Johnston DG (1966) Blood oxygen and ecology of porpoises of three genera. Science 151:456–457 Salathe EP, Chen C (1993) The role of myoglobin in retarding oxygen depletion in skeletal muscle. Math Biosci 116:1–20 Saunders DK, Fedde MR (1991) Physical conditioning: effect on the myoglobin concentration in skeletal and cardiac muscle of barheaded geese. Comp Biochem Physiol A 100:349–352 Schreer JF, Kovacs KM, O’Hara Hines RJ (2001) Comparative diving patterns of pinnipeds and seabirds. Ecol Monogr 71(1):137–162 Stephenson R, Turner DL, Butler PJ (1989) The relationship between diving activity and oxygen storage capacity in the tufted duck (Aythya fuligula). J Exp Biol 141:265–275 Thorson PH (1993) Development of diving in the northern elephant seal. Ph.D. diss., University of California, Santa Cruz Velten BP (2012) A comparative study of the locomotor muscle of extreme deep-diving-Cetaceans. M.S., University of North Carolina, Wilmington Velten BP, Dillaman RM, Kinsey ST, McLellan WA, Pabst DA (2013) Novel locomotor muscle design in extreme deep-diving whales. J Exp Biol 216:1862–1871 Verrier D, Guinet C, Authier M, Tremblay Y, Shaffer S, Costa DP, Groscolas R, Arnould JPY (2011) The ontogeny of diving abilities in subantarctic fur seal pups: developmental trade-off in response to extreme fasting? Funct Ecol 25(4):818–828 Weber RE, Hemmingsen EA, Johansen K (1974) Functional and biochemical studies of penguin myoglobin. Comp Biochem Physiol B 49:197–214 Weise MJ, Costa DP (2007) Total body oxygen stores and physiological diving capacity of California sea lions as a function of sex and age. J Exp Biol 210:278–289 Wells RS (1991) Bringing up baby. Nat Hist 100(8):56–62 Westgate AJ, Read AJ, Berggren P, Koopman HN, Gaskin DE (1995) Diving behaviour of harbour porpoise, Phocoena phocoena. Can J Fish Aquat Sci 52:1064–1073 Williams TM, Noren SR, Glenn M (2011) Extreme physiological adaptations as predictors of climate-change sensitivity in the narwhal, Monodon monoceros. Mar Mamm Sci 27(2):334–349