1793
The Journal of Experimental Biology 216, 1793-1798
© 2013. Published by The Company of Biologists Ltd
doi:10.1242/jeb.082099
RESEARCH ARTICLE
Rapid postnatal development of myoglobin from large liver iron stores in
hooded seals
Samuel J. Geiseler1,*, Arnoldus S. Blix1, Jennifer M. Burns2 and Lars P. Folkow1
1
Department of Arctic and Marine Biology, University of Tromsø, NO-9037 Tromsø, Norway and 2Department of Biological Sciences,
University of Alaska Anchorage, Anchorage, AK 99508, USA
*Author for correspondence ([email protected])
SUMMARY
Hooded seals (Cystophora cristata) rely on large stores of oxygen, either bound to hemoglobin or myoglobin (Mb), to support
prolonged diving activity. Pups are born with fully developed hemoglobin stores, but their Mb levels are only 25–30% of adult
levels. We measured changes in muscle [Mb] from birth to 1year of age in two groups of captive hooded seal pups, one being
maintained in a seawater pool and one on land during the first 2months. All pups fasted during the first month, but were fed from
then on. The [Mb] of the swimming muscle musculus longissimus dorsi (LD) doubled during the month of fasting in the pool
group. These animals had significantly higher levels and a more rapid rise in LD [Mb] than those kept on land. The [Mb] of the
shoulder muscle, m. supraspinatus, which is less active in both swimming and hauled-out animals, was consistently lower than
in the LD and did not differ between groups. This suggests that a major part of the postnatal rise in LD [Mb] is triggered by
(swimming) activity, and this coincides with the previously reported rapid early development of diving capacity in wild hooded
seal pups. Liver iron concentration, as determined from another 25 hooded seals of various ages, was almost 10 times higher in
young pups (1–34days) than in yearling animals and adults, and liver iron content of pups dropped during the first month,
implying that liver iron stores support the rapid initial rise in [Mb].
Key words: diving, neonate, activity, liver iron content, Cystophora cristata.
Received 25 October 2012; Accepted 19 January 2013
INTRODUCTION
Seals and whales depend on oxygen stores bound to hemoglobin
(Hb) in the blood and to myoglobin (Mb) in skeletal muscles to
support aerobic metabolism during diving, and their dive capacity
is, accordingly, positively correlated with blood volume, blood [Hb]
and muscle [Mb] (Mottishaw et al., 1999; Noren and Williams,
2000).
Adult hooded seals (Cystophora cristata) have the largest oxygenstoring capacity per unit body mass of any mammal hitherto
examined (Burns et al., 2007; Lestyk et al., 2009), and may stay
submerged for more than 1h (Folkow and Blix, 1999). Newborn
hooded seals, in contrast, have a mass-specific blood oxygen-storing
capacity that is similar to that of adults, but their muscle [Mb] is
only about 25% of the adult value (Burns et al., 2007). Even so,
these pups, which have the shortest lactation period of any mammal
(Bowen et al., 1985), also have a very rapid development of diving
capacity, and may dive to depths of >100m for durations of >15min
before reaching 3weeks of age (Folkow et al., 2010).
The development of oxygen storage capacity is known to involve
hypoxia-inducible factor-1 (HIF-1), which among other things
regulates the expression of erythropoietin and hence synthesis of
Hb (Gassmann and Wenger, 1997). The seal fetus is repeatedly
exposed to hypoxia in utero, when the mother is diving (Elsner et
al., 1969; Liggins et al., 1980), and this most likely triggers the
expression of HIF-1 and the development of blood O2 stores before
birth. Mb expression, however, has been shown to be regulated by
the calcium–calcineurin–NFAT (nuclear factor of activated T-cells)
pathway (Bassel-Duby et al., 1993; Kanatous et al., 2009), which
in turn is also partly triggered by hypoxia, but apparently only in
combination with muscular activity, at least in mice (Kanatous et
al., 2009; Wittenberg, 2009). A recent study using cultured myocytes
from Weddell seals (Leptonychotes weddellii) showed that control
of Mb development in these animals differs somewhat from that
outlined above, in that Mb levels were found to increase in response
to hypoxia (and also after lipid supplementation), even in the absence
of contraction (De Miranda et al., 2012). The authors nevertheless
concluded that a secondary stimulus would still be required to
increase Mb levels to those seen in the whole animal. As movement
is rather restricted for the fetus, muscular activity may be the trigger
that is missing for full Mb development in utero in the hooded seal.
For Mb development to occur, some source of iron would be
required, given this element is a crucial component of this and
other heme proteins. As hooded seal pups may undergo an
extended fasting period after their very brief nursing period (e.g.
Bowen et al., 1987; Oftedal et al., 1989), any Mb synthesis in
this period must be based on endogenous stores of iron. The major
compartments for iron storage in mammals are the Hb pool and
the liver (e.g. Fleming and Bacon, 2005; Graham et al., 2007).
As it would be counter-productive to catabolize Hb in order to
obtain iron for Mb synthesis, we hypothesized that any Mb
development that takes place during fasting in these pups is likely
to primarily be supported by mobilization of ferritin-bound iron
from their liver.
The present study, thus, had a dual purpose: first, to describe the
early postnatal development of the myoglobin stores in hooded seal
pups and investigate whether muscular activity affects this
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1794 The Journal of Experimental Biology 216 (10)
development; and second, to determine whether their liver is a likely
source of iron for postnatal Mb development.
MATERIALS AND METHODS
Animals
A total of 33 hooded seals, C. cristata (Erxleben 1777), were used
in this study. Eight newly weaned pups (aged 4–6days, as judged
from external characteristics and the presence/absence of a mother)
(Bowen et al., 1987) were collected in the pack ice of the Greenland
Sea (the West-ice, at about 71°40′N, 14°20′W) between 22 and 27
March 2010 during a research cruise with R/V Jan Mayen, under
permits from the Norwegian and Greenland authorities. These pups
were brought to Tromsø, Norway, where they were kept in the
approved research animal facility of the Department of Arctic and
Marine Biology at the University of Tromsø, for studies of postnatal
changes in muscle Mb levels. The animals were divided into two
groups: four animals were kept in a 40,000l indoor seawater pool
that permitted swimming activity (pool group), while the remaining
four pups were kept in a fenced snow-covered ~25m2 outdoor
enclosure with no possibility of swimming (land group). After
~2months (age of pups ~70days), when conditions no longer
allowed the latter to be maintained outdoors because of snow-melt,
the land group was transferred indoors to another 40,000l seawater
pool, which was identical and adjacent to the one already occupied
by the pool group. In both pools, a wooden ledge allowed the seals
to haul out as desired. To mirror the naturally occurring 1month
post-weaning fast (Bowen et al., 1987), captive pups were not fed
until the age of ~37days, after which they were fed capelin
(Mallotus villosus) and subsequently herring (Clupea harengus).
Dietary intake of fish gradually increased from a few fish per day
to 2.0–2.5kg of fish per day by the time pups were ~2months old
and onwards. The diet was supplemented with a vitamin complex
(Sea Tabs II for marine mammals, Pacific Research Laboratories,
CA, USA). All animals were subjected to normal light/dark cycles
at 69°N latitude, either natural (outdoor land group) or simulated
(indoor pool group).
Twenty-one other hooded seals (12 pups, aged 1–10days; four
yearlings; and five adults, aged 2–16years) were culled in the same
general area (~72°00′N, 16°40′W) in which the eight pups were
caught, during similar research cruises to the Greenland Sea in March
2011 and 2012, for collection of miscellaneous data and tissue
samples for a range of scientific purposes, including liver mass and
samples for analyses of liver iron contents for the present study.
Additional liver mass data were obtained from our own unpublished
records (L.P.F., unpublished data) and from published data (Oftedal
et al., 1989). The pups were aged from external characteristics and
the presence/absence of a mother (Bowen et al., 1987), yearlings
from external characteristics, and adults based on inspection of
sectioned teeth under the microscope as described elsewhere
(Rasmussen, 1960).
Four newly weaned pups were captured during the 2011
research cruise to the Greenland Sea and transferred to Tromsø
where they were kept under conditions that were identical with
those of the pool group in 2010, i.e. including a 30day fasting
period (see above). These pups were killed at the age of ~34days
for collection of various samples, including liver samples for
analysis of iron content.
All animals were killed in accordance with the Norwegian Animal
Welfare Act, through stunning [shot through the head or given an
overdose of pentobarbital (Nembutal, 20mgkg−1) injected into the
extradural intravertebral vein] immediately followed by bleeding
through severing of the brachial vasculature. All use of research
animals was approved by the National Animal Research Authority
of Norway (permit no. 2402).
Muscle, blood and liver sampling
Muscle biopsies were collected at intervals from the main swimming
muscle (musculus longissimus dorsi, LD) and from one of the flipper
muscles (musculus supraspinatus, SSP) of the eight pups that were
captured in 2010. The pups were first sampled when about 1week
old, while the last biopsies were collected when they were ~1year
of age (394days). The sampling interval during the first month was
10days, which was then extended to every 2weeks during the second
month, and was bi-monthly from there on. Prior to sampling, animals
were weighed (model 235 suspended weight; Salter, Tonbridge,
Kent, UK) and then sedated with an intramuscular injection of Zoletil
Forte Vet (1–1.5mgkg−1; tiletamin–zolazepam, Virbac, Carros
Cedex, France) supplemented when needed with i.v. injections
(0.2–0.3mgkg−1) via a venous catheter (Secalon T,
IGG/1.7×160mm; Becton Dickinson, Franklin Lakes, NJ, USA) in
the extradural intravertebral vein. Muscle biopsies were collected
under additional local anesthesia, after injection of Xylocaine
(3–4ml of 10mgml−1; Astra Zeneca, Södertälje, Sweden) in the
incision region. The skin area (~3×3cm) was shaved and disinfected
with chlorhexidine (5mgml−1; Fresenius Kabi, Halden, Norway).
A small incision was then made with a sterile scalpel (blade no. 11)
and the sample was collected using a sterile disposable 6mm biopsy
punch (Miltex, York, PA, USA) that was advanced through the skin
and blubber into the underlying muscle. Collected (duplicate)
samples were temporarily stored on ice and then transferred to
cryovials and frozen at −80°C for later analysis. After sampling,
the incision site was closed with absorbable suture and the animals
returned to their holding facilities immediately after recovery from
sedation. Sites for sampling were alternated between the two body
sides, with LD sampling sites being located in the upper lumbar
region. On each sampling occasion, a blood sample was also
collected via the central venous catheter in the extradural
intravertebral vein, for determination of hematocrit (Hct) values by
use of a hematocrit centrifuge (EBA 12, Type 1000, Hettich,
Tuttlingen, Germany).
Liver samples were collected from the 25 culled animals of
various ages (Table1), by cutting a central piece (~4cm3) from the
biggest of the six liver lobes. The samples were frozen at −20°C
for later analysis of iron content.
Mb analyses
[Mb] was determined according to Reynafarje (Reynafarje, 1963),
as previously described (Burns et al., 2007; Lestyk et al., 2009).
Briefly, frozen muscle samples were thawed, cleaned of connective
tissue and blood and sonicated (Sonic Dismembrator model 500,
Fisher Scientific, Fairlawn, NJ, USA) in ice-cold 0.04moll–1
phosphate buffer (19.25mlg−1 tissue, pH6.6). The samples were
then centrifuged at 10,000g for 5min at 4°C. The supernatant was
placed in a vacuum chamber, which was first gassed with CO
(99.5%) for 30s, then filled with CO for 15s and closed. After 20min
of CO incubation, sodium dithionite solution was added (1% in
sample) and the samples were vortexed to ensure full reduction of
Mb for correct absorption measurement. After an additional 5min
of CO incubation, the optical density was read at 538 and 568nm
(Spectra Max 340PC, Molecular Devices, Sunnyvale, CA, USA)
and [Mb] was calculated.
Assays were run in triplicate and each run included both
lyophilized Mb (Sigma, St Louis, MO, USA) and tissue controls
from an adult harbor seal (Phoca vitulina) with known Mb levels
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Myoglobin synthesis in hooded seal pups
1795
Table 1. Mean (±s.d.) body and liver mass, liver mass as percentage of body mass, wet liver iron concentration and estimated mean
total liver iron stores (total and mass-specific) in hooded seals of various age classes
Age class (age)
Newborn–weaned
(1–10 days)
Fasted pups
(~34 days)
Yearling (1 year)
Adults (2 year)
Body mass (kg)
Liver mass (g)
Liver mass
(% body mass)
33.0±10.29
(23.0–62.5)
N=19
32.2±1.19
(27.8–35.5)
N=4
61.50
(53.0–70.0)
N=2
143.1±29.66
(94.0–180.0)
N=7
953±359.5
(410–1970)
N=19
511±19.0*
2.92±0.692
(1.64–4.04)
N=19
1.59±0.057*
1340
(1010–1670)
N=2
3177±802.7
(2256–4629)
N=7
2.18
(1.91–2.39)
N=2
2.22±0.261
(1.92–2.57)
N=7
Liver [Fe]
(μg g1)
889±467
(249–1782)
N=13
1301±572
(586–1949)
N=4
113±100
(61–263)
N=4
161±84
(75–291)
N=5
Estimated mean
total liver Fe
(mg)
Estimated mean
mass-specific liver
Fe (mg kg1)
847
25.7
665*
20.6*
151
2.5
511
3.6
Ranges are given below mean ± s.d. data in parentheses.
*Values are based on data for liver mass of N=36 hooded seals pups of similar mass (31.4±0.98 kg, mean ± s.e.m.) after 1 month of the post-suckling fast
(Oftedal et al.,1989).
(Burns et al., 2007), to validate the results. To estimate the variance
within the muscles, three large samples of LD and SSP muscles,
respectively, were collected from one of the culled animals and
analyzed. The precision of the assay was estimated from the harbor
seal tissue controls.
Liver iron analyses
Non-heme liver iron content was determined as described previously
(Rebouche et al., 2004), as non-heme iron concentration per tissue
wet mass. Briefly, liver samples were thawed and homogenized in
high-purity water (1:10 w/v) and protein precipitation solution
[1moll–1 HCl and 10% trichloroacetic acid (Fisher Scientific) in
high-purity water] was added (1:1). After incubation for 1h at 95°C,
the samples were vortexed and centrifuged at 8200g for 10min at
room temperature. The supernatant was mixed (1:1) with chromagen
solution [0.508mmoll−1 ferrozine (Sigma), 1.5moll–1 sodium acetate
(Fisher Scientific) and 0.1% or 1.5% (v/v) thioglycolic acid (Sigma)
in high-purity water]. After 30min incubation at room temperature,
the samples were centrifuged at 8200g and the optical density of
the supernatant (triplicates) read at 562nm (Spectra Max 340PC).
An iron standard (Sigma) and sample blanks were prepared by
mixing the supernatant with chromagen solution without ferrozine.
Liver water content was determined by drying separate samples from
each age class for 48h at 70°C. Data on liver masses were used to
estimate liver iron content.
Data handling and statistics
A linear mixed model approach was used to analyse the Mb data.
To determine the effect of muscle type, group location and age on
Mb, each of these parameters was tested in a full model that included
other parameters as fixed factors. If the muscle type had a significant
effect, i.e. there was a significant difference between the muscle
types, the data were split and analysed for the two types separately.
If group location had a significant effect, the data were further split
and analysed for each group, to determine the effect of time. In
addition, a pair-wise comparison of the different age classes was
conducted. If a parameter had no significant effect, it was removed
from the model. To determine the effect of group location for each
age class (i.e. at each sampling occasion), the data were split to
analyse each separately. Between-groups comparisons were only
made until age ~70days, as after that time both the pool and the
land groups were maintained in (separate) pools. Pairwise
comparisons were adjusted for multiple comparisons with the
Bonferroni method.
Age-wise comparisons of liver iron concentration and content
were made using independent samples t-tests.
For all tests, P<0.05 was considered significant. Values are
presented as means ± s.d. unless otherwise stated. All statistical
analyses were made using SPSS v.19.0 (SPSS Inc., Chicago, IL,
USA).
RESULTS
The body mass of the eight pups that were kept in Tromsø for
collection of muscle biopsies dropped by ~30% from 44.2±3.8kg
to 32.3±2.2kg during the 30day fasting period and thereafter
increased to 68–85kg at 1year of age (Fig.1A). There was no
significant difference in body mass between the two groups
(F1,70=1.250, P=0.267). At the age of ~70days, the land group was
reduced from four to three animals, as a result of the death of one
pup during sedation.
Hct values were similar in the two groups and remained stable
at around 60% (overall mean Hctpool=58.87±4.38%,
Hctland=59.29±4.35%) throughout the period (Fig.1B).
There was a significant difference in LD [Mb] between the pool
and land group (F1,35=10.9, P=0.002, mean difference
pool–land=5.91±1.79mg Mbg−1), with pool animals having higher
levels than land animals (Fig.2). For both groups, LD [Mb] showed
a significant increase with age (pool: F5,18=6.878, P=0.001; land:
F5,17=4.912, P=0.006), until 65days, but then leveled off (Fig.2).
With regard to SSP, there was no statistically significant
difference in Mb levels between the two groups. Moreover, age had
a significant influence on the SSP Mb values only in the pool group
(F6,17=4.077, P=0.013). Mean Mb levels were significantly lower
in SSP than in LD in both groups and at all ages (pool: F1,35=23.883,
P<0.001; land: F1,34=4.434, P=0.043), but the mean difference was
higher in the pool group than in the land group (pool:
LD–SSP=7.95±1.63mg Mbg−1; land: LD–SSP=3.58±1.70mg
Mbg−1. The SSP Mb levels, like the LD Mb levels, rose steadily at
the beginning of the sampling period, but then leveled off (estimated
variation within the muscles: CVLD=5.7%, CVSSP=6.1%; estimated
precision of the assays: 95%).
Data on body mass, liver mass and liver iron concentration of
the seals that were used for liver iron content analyses are shown
in Table1. Liver mass on average corresponded to 2.22% of adult
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1796 The Journal of Experimental Biology 216 (10)
Body mass (kg)
80
70
A
70
60
50
40
30
*
Pool
Land
Newborn
60
[Mb] (mg g–1 tissue)
90
*
(4)
*
(3)
(4)
(4)(4)
50
(4)
(4)
40
(4)
(4)
(3)
(4)
(4)
(8)
30
20
20
0
100
200
300
400
10
7
100
B
80
Hct (%)
(8) (8)
(7)
(7)
(7)
(7)
60
17
26
37
68
Age (days)
94
394
Fig.2. Temporal changes in myoglobin (Mb) concentration (means ± s.d.)
of the m. longissimus dorsi (LD) in the two groups of captive hooded seal
pups described in Fig.1 and in Materials and methods. Numbers in
parentheses above the bars are the number of seals. Newborn data are
pooled data for all animals before separation into groups. *Significant
differences in Mb levels between groups.
40
20
DISCUSSION
0
0
100
200
300
400
Age (days)
Fig.1. (A)Temporal changes in body mass (means ± s.d.) of the eight
hooded seal pups that were maintained in captivity in two groups for
~400days. The pups were not fed until the age of ~34days. (B)Temporal
changes in hematocrit (Hct) values (means ± s.d.) of the same animals as
in A. Numbers in parentheses above the bars are the number of seals.
Data on mass and Hct were pooled as there were no significant differences
between the two groups.
body mass and 2.18% of yearling body mass, which was lower than
that in young pups (2.92%), but higher than that in the fasted pups
(1.59%) (Oftedal et al., 1989). There was no significant difference
in liver mass within the young pup age class even though weaned
pups tended to have slightly higher values (newborn: 843±453g,
N=10; weaned: 1114±433g, N=9).
Liver iron concentration displayed large variance, even within
age classes. There were no significant differences in liver iron
concentration among pups aged 1–34days (F=0.043, P=0.838),
although fasted pups had somewhat higher concentrations than
young pups (Table1). There were no significant differences in liver
iron concentration among older (>1year) animals (F=0.152,
P=0.708). Overall, pups (1–34days) had significantly higher liver
iron concentrations than older animals (986±508 versus
140±89μgg–1 wet mass, F=19.69, P<0.001) (Fig.3).
The estimated mean total liver iron content was highest
immediately after birth, in both absolute and relative (mass-specific)
terms (Table1). During the fasting period (during which LD Mb
levels doubled), liver iron content decreased by 25%, but was still
higher than in yearlings, which also had had access to dietary iron
to sustain development of Hb and Mb levels during growth.
The liver water content ranged between 60.0 and 63.6% in
newborns (1–10days) and 69.1 and 71.6% in the older animals
(34days to 16years).
Blood Hct was stable at the adult level (~60%) throughout this study
(Fig.1B), which confirms that hooded seals have fully developed
blood oxygen stores from the very beginning of life (Burns et al.,
2007).
However, we have shown for the first time that while [Mb] is
low at birth, there is a rapid initial increase in [Mb] in the LD muscle
of hooded seal pups, from 25% to 50% of adult values, within their
first month of life (Fig.2). It is intriguing that this remarkable rise
in [Mb] coincides closely with the development of diving capacity
in these animals (Fig.4) (Folkow et al., 2010).
We further conclude that this postnatal rapid rise in [Mb] was
mainly due to increased muscular activity. We base this conclusion
on the following arguments. The pool group, which indulged in
vigorous swimming, known to involve the LD muscles (Kanatous
et al., 1999), both showed a more rapid development of LD [Mb]
and maintained significantly higher [Mb] at all times than did the
LD muscle of animals in the land group (Fig.2), which typically
were immobile for most of the time and certainly did not engage
in swimming or other activities that involved the LD muscles. We
further note that [Mb] in the SSP muscles, which apparently were
not used to any particular extent either in the pool or in the land
group, was both significantly lower than in the LD muscle
(regardless of group) and quite similar between the two groups.
If we, for the sake of the argument, assume that (diving-induced)
hypoxia, instead of muscular activity, is the main trigger for Mb
development, we would expect between-groups differences in Mb
levels for both muscle types (i.e. also for the SSP muscles), but this
was not the case. It could be argued that the dives of the pool group
animals were not of sufficient duration (typically 1–2min) to produce
any substantial hypoxia, and that land group animals might have
experienced equivalent hypoxia in connection with sleep apnea, but
in that case there is no good reason why LD Mb levels should differ
between the groups – unless Mb development requires muscular
activity, as we have concluded. Thus, the mere fact that [Mb]
increased at a similar rate in the SSP but at a different rate in the
LD in the two groups suggests that activity, at least, is a crucial
component for the development of Mb, which is in agreement with
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Myoglobin synthesis in hooded seal pups
10
(17)
1400
A
8
1200
1000
Idur (min)
Liver [Fe] (µg g–1 wet mass)
1600
1797
800
600
6
4
400
(9)
2
200
0
Young
0
Old
0
Fig.3. Liver iron concentration (means ± s.d.) in two age categories of
hooded seals: young (1–34days) and old (1–16years). The young age
class had significantly higher values than the older animals (986±508
versus 140±89μgg−1 wet mass, F=19.69, P<0.001). Numbers in
parentheses above the bars are the number of seals.
[Mb] (mg g–1 tissue)
findings in other species (Pattengale and Holloszy, 1967; Kanatous
et al., 2009; Wittenberg, 2009).
Our captive animals had lower [Mb] at 1year of age than wild
animals (Burns et al., 2007), which is probably due to reduced
activity and a much lower exposure to hypoxia in captivity. Similar
observations and conclusions have been made for other diving
species (e.g. emperor penguins, Aptenodytes forsteri) (Ponganis et
al., 2010). This implies that wild animals probably show an even
more remarkable initial rise in [Mb] than our captive pups.
It is worth noting that a significant part of the rapid postnatal rise
in [Mb] occurred while our pups were fasting, which happens naturally
at that time (Bowen et al., 1987). As blood Hct values remained stable,
we also assume that no catabolism of blood Hb took place that could
make iron available for Mb synthesis. We hypothesize that the iron
required for this rapid production of Mb must have been derived from
other internal stores, such as the liver. This organ is known to represent
a key compartment for storage of iron in humans and other species,
in which dietary iron is taken up from the portal blood, and, at times
of increased demand, released into the circulation (e.g. Fleming and
Bacon, 2005; Graham et al., 2007). This hypothesis is supported by
our observation of significantly higher liver iron concentrations (and
content) in pups than in older animals (Fig.3). Also, although fasted
pups apparently had higher liver iron concentrations than did newborn
pups (but this difference was non-significant), their liver mass was
only about half that of the newborns, which means that liver iron
content decreased substantially during the 1month of fasting (Table1).
The reason why the hooded seal liver undergoes this large mass change
during fasting is not known, but it probably relates to mobilization
of large liver-based glycogen stores during the early part of the fast,
as previously shown in other fasting/starving mammals (e.g. Nilsson
and Hultman, 1973).
If we assume that blubber was the main source of energy during
the postnatal fasting period and that muscle mass thereby remained
largely unchanged, as in grey seal (Halichoerus grypus) pups
(Nordøy and Blix, 1985), the amount of Mb produced may be
estimated based on data from Burns and associates (Burns et al.,
2007), which show that the skeletal muscle mass of a weanling
hooded seal corresponds to about 20% of its body mass, or 8.8kg
in a 44.2kg pup (which was the average mass of our eight weanlings;
Fig.1A). With an average increase in [Mb] in this muscle mass from
~25 to ~35mgg−1 within the fasting period (Fig.4B), about 88g of
50
20
40
60
80
100
B
40
30
20
0
20
40
60
80
100
Age (days)
Fig.4. (A)Changes in diving duration indices (Idur) with age in seven
hooded seal pups that were tracked with satellite-linked dive recorders in
2006–2009 (modified from Folkow et al., 2010). Idur represents a weighted
estimate of the average duration of individual dives by the pups and was
calculated as described in detail elsewhere (Folkow et al., 2010). Day 0
corresponds to 27 March, which was also the approximate date of birth of
the eight captive pups in B. Diving duration increased rapidly during the
first 40days and then leveled off. (B)Changes in mean (and s.d.) LD and
musculus supraspinatus (SSP) Mb levels combined, of seven to eight
hooded seal pups (data for pool and land groups have been combined).
The Mb levels increased significantly over the first 30–40days and then
leveled off.
Mb may have been synthesized, which would require mobilization
of 280mg of iron (given the need for one Fe for each Mb molecule
and a molecular weight ratio for Fe/Mb of 55.84/17,380). The
estimated decrease in liver iron content in the same period was about
182mg (Table1), which is fairly close to the estimated iron required
for Mb synthesis, particularly when considering the remarkable
variation in the liver iron concentration in seals, as in other species
(Zuyderhoudt et al., 1978; Faa et al., 1994; Bonkovsky et al., 1999).
In estimating liver iron content in young pups (newborn/weaned;
Table1), we have pooled the data on liver mass and liver iron
concentration for all pups aged 1–10days, as there was no significant
difference in the liver data within this age group. Moreover, hooded
seal pups are physically inactive during the suckling period, which
is largely spent suckling or sleeping. Although milk-derived lipids
may have stimulated some Mb development (Burns et al., 2010; De
Miranda et al., 2012), we do not expect that much liver iron was
mobilized for Mb synthesis during lactation, as this period lasts for
only about 4days in this species (Bowen et al., 1987).
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1798 The Journal of Experimental Biology 216 (10)
It may appear puzzling that Mb, unlike Hb, is not well developed
from birth in these soon-to-become expert diving seals, as fully
developed Mb stores would allow a more rapid development of diving
capacity. However, pregnant seal mothers have been shown to
continue their normal diving pattern until shortly before delivery
(Liggins et al., 1980) and it has been shown indirectly that the fetus,
unlike the mother, depends on chemoreceptor stimulation to develop
the cardiovascular defense responses to hypoxia (Elsner et al., 1969).
This implies that the instant selective vasoconstriction and bradycardia
that are characteristic of adult animals in dives of long duration (Blix
and Folkow, 1983; Ramirez et al., 2007) take time to develop in the
fetus. Moreover, as Mb has a much higher affinity for oxygen than
does Hb, a high concentration of Mb would, with no benefit to the
immobile fetus, deplete the blood of oxygen, particularly at the end
of dives, when maternal arterial oxygen tension sometimes reaches
extremely low levels. It therefore appears to be more advantageous
to build Mb stores at a rapid rate after birth, as seems to be the case
in hooded seal pups, rather than have high levels from birth.
ACKNOWLEDGEMENTS
We thank Professor E. S. Nordøy and the crew of R/V Jan Mayen for assistance
in the field, and Dr Chandra Ravuri for laboratory assistance.
AUTHOR CONTRIBUTIONS
S.J.G., A.S.B., J.M.B. and L.P.F. were all involved in the conception, design and
execution of the study, the interpretation of findings, and drafting and revising the
article.
COMPETING INTERESTS
No competing interests declared.
FUNDING
S.J.G., A.S.B. and L.P.F. were supported by faculty grants from the Department of
Arctic and Marine Biology, University of Tromsø, and J.M.B. was supported by a
faculty grant from the College of Arts and Sciences, University of Alaska,
Anchorage.
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