Assessing a relationship between bone microstructure and growth rate: a fluorescent labelling study in the king penguin chick (Aptenodytes patagonicus)
1 Adaptation et Evolution des Systèmes Ostéo-Musculaires, FRE
CNRS 2696, 2 place Jussieu, 75251 Paris Cedex 05, France
2 Centre d'Ecologie et Physiologie Energétiques, UPR CNRS 9010, 23
rue Becquerel, 67087 Strasbourg Cedex 2, France
* Author for correspondence (e-mail: margerie.e.de{at}club-internet.fr)
Accepted 15 December 2003
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Summary |
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Key words: fibro-lamellar bone tissue, biomechanical properties, bone microstructure, growth rate, long bone, structurefunction
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Introduction |
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King penguins are flightless seabirds with a unique growth strategy
(Stonehouse, 1960;
Barrat, 1976
): between hatching
and fledging (i.e. departure to sea), chicks spend almost one year on land in
the breeding colony. They experience a first phase of rapid growth
(approximately 3 months between February and early May). During this phase,
chicks are intensively fed by their parents; they achieve a 4060-fold
increase in body mass (from 0.2 kg to 912 kg) and almost reach the
adult size, which is among the largest in extant neognathe birds (1 m tall,
12 kg; Prévost and Mougin,
1970
; Barrat,
1976
). A period of very low feeding rate follows, during the
austral winter (4.5 months, May to mid-September). Chicks lose half of their
body mass, and only chicks that have large initial body energy reserves have a
high survival rate (Barrat,
1976
; Cherel et al.,
1987
). The last 3.5 months in the colony (mid-September to late
December) start with the recovery of an intensive parental feeding rate, see
ponderal recovery of chicks and end with chick moulting, immediately followed
by their departure to open sea.
Our study focused on the first very active growth phase.
Chicks were studied between their third and fifth week, i.e. when the growth
rate of their flipper and leg bones was the highest
(Stonehouse, 1960). Bone
tissue growth rates were measured in the appendicular skeleton, coupled with
the investigation of associated bone tissue types, which yielded appropriate
data to test experimentally Amprino's theory
(Amprino, 1947
).
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Materials and methods |
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During these first weeks of life, chicks were either always with a parent
or, during the time (510 min) a chick was manipulated in a nearby
shelter for injections and body measurements, were exchanged with a dummy egg,
a procedure well accepted by the parents. Until the chick was returned, the
`incubating' parent was continuously observed. Each time they were
manipulated, birds were weighed (±10 g) and their bill, flipper and
foot lengths measured (±0.5 mm). Between each weekly manipulation,
chicks were left undisturbed in the breeding colony with their parents and
were observed daily. They were identified with coloured fish-tags implanted
dorsally in the skin under local anaesthesia (1 ml xylocaine 2%) at the first
manipulation. Each chick's parents were identified through dye marks on the
chest. The growth curve (body mass and body measurements) of the four labelled
chicks was compared with that of 30 unlabelled chicks born in the same area of
the colony at similar dates (identified in the same manner as labelled
chicks).
The labelled chicks were sacrificed by cervical vertebrae dislocation after being emancipated (i.e. left alone in the colony by their parents between two feedings) when they were 5 weeks old (except for one chick, who died accidentally during a storm 4 days after the third labelling and suffered partly from predation). They were kept frozen (20°C) until further analysis.
The authorisation to perform these experiments in the breeding colony and to sacrifice chicks was obtained from the Ethics Committee of the Institut Français pour la Recherche et la Technologie Polaire (present name Institut Polaire Paul-Emile Victor). The study followed the `Agreed Measures for the Conservation of Antarctic and Subantarctic Fauna'.
Bone sections
Four long bones (humerus, radius, femur and tibiotarsus;
Fig. 1) were removed per
individual and embedded in polyester resin, after dehydration in graded
ethanol and defatting in acetone and trichloroethylene. A 500 µm-thick
mid-diaphyseal cross section was sawed out of each bone using a Presi P-100
diamond saw (Grenoble, France). Sections were glued to a glass object-holder
with epoxy glue (Devcon, Riviera Beach, FL, USA) and ground to 80 µm using
graded abrasive material.
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Measurements of growth rates
Periosteal growth rates were measured optically under ultraviolet light on
a fluorescence microscope (Zeiss Axiovert 35; Jena, Germany; magnification
50x), using the following method. Each section was observed in four
orthogonal anatomical directions (e.g. anterior, posterior, dorsal and
ventral). In each field, three successive fluorescent labels and the
periosteum delimited three bone tissue `patches' (examples in
Fig. 2), corresponding to week
3, week 4 or week 5 in the chick's growth. Each patch was characterised by a
bone tissue type, identified under natural light, following de Ricqlès'
classification of bone tissues (de
Ricqlès, 1975; de
Ricqlès et al., 1991
). Using an optical micrometer, the
thickness of each patch was measured once (±10 µm) as the distance
between the outer borders of labels (Fig.
2) and then divided by the time elapsed between two labels to
yield a local value of growth rate. Out of a theoretical sum of 192 (three
patches per direction, four directions per section, four sections per chick,
four chicks), we were unable to analyse 52 patches because, occasionally, the
first (and even second) label was locally destroyed by perimedullar
resorption. Moreover, the periosteum was sometimes difficult to locate
precisely, invalidating some measures of week 5. The data from the 140
analysed patches are presented in Table
1.
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Statistical analysis
We analysed our data using the following analysis of variance (ANOVA)
design: growth rate (the quantitative variable) was considered as the
dependent variable. Growth rates were log-transformed to satisfy the
`homogeneity of variance' assumption of parametric ANOVA. This transformation
also conferred a normal distribution to the 140 growth rate values. In
agreement with the histological level of integration of our study, bone tissue
patches were the statistical elements of the design (and not the four chicks,
which constitute a grouping variable here). Four fixed factors (categorical
grouping variables) were assumed to have a possible effect on growth rate:
Unfortunately, as a consequence of working on limited and precious material, our data were insufficient to test the effects of all factors and interactions simultaneously in a complete controlled four-way ANOVA design. Indeed, our data set contains too few replications of measurements per cell in the table and a substantial amount of missing data. To circumvent this restriction, we first tested the difference between chicks and between weeks of growth using one-way ANOVAs. Since these tests returned no significant effects of the two factors (see Table 2), we pooled growth rate data across chicks and weeks of growth. This reduced the number of cells in the data set and therefore increased replication of measurements within each cell. This enabled us to test the effects of the two remaining factors (long bone and bone tissue type, which are of main interest here) in a complete controlled two-way ANOVA design. Post-hoc multiple comparisons were carried out using HSD Tukey test for unequal replication (5% experiment-wise error rate). They allowed groupings of non-significantly different levels within each factor.
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Body mass and measurements at the beginning and end of the experiment were compared using paired t-tests. Ages of emancipation between labelled and non-labelled chicks were compared using t-test. Growth curves of labelled and non-labelled chicks (Fig. 3) were compared using the 95% confidence intervals of their linear slopes.
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STATISTICA and STATVIEW software were used to perform the analysis.
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Results |
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Bone microstructure
Each bone section comprised two concentric regions: a peripheral crown, or
`cortex', encircling a central area of lesser compactness, the `medulla'
(Fig. 1).
Cortex
Five-week-old chicks had bone cortices made of fine cancellous bone
(Fig. 1). This structure
results from periosteal growth of `fibro-lamellar bone', the rapidly growing
type of bone tissue (see de Ricqlès
et al., 1991). Fluorescent labels
(Fig. 2) demonstrated rapid
periosteal bone growth, i.e. apposition of a highly lacunar woven network at
the bone periphery, completed by a progressive filling by primary osteons in
the depth of the cortex. Such fine porosity of the whole cortex is due to fast
centrifugal progression of the periosteum, exceeding the primary osteon
filling process, which thus remains temporarily incomplete. Primary osteon
orientation was quite variable, even within a single cross section.
Accordingly, four fibro-lamellar bone tissue types could be distinguished:
All four tissue types were common, exhibited by every bone of our study, with various frequencies (Fig. 4).
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Finally, the chicks' cortices were thick, occupying the outer third of the shaft's radius, on average. This high cortical thickness is the result of limited perimedullar resorption (osteoclasts progressing centrifugally at the inner surface of the cortex), which proceeds slowly and/or starts late compared with periosteal apposition.
Medulla
A rather loose medullary spongiosa occupied the central area of the
sections (Fig. 1). It was made
of remnants of cortical primary bone, spared by perimedullar resorption. These
trabeculae had been remodelled and consolidated by some endosteal bone
apposition, as shown by fluorescent labels
(Fig. 5).
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Periosteal bone growth rate
The 140 growth rate values ranged from 7 µm day1 to
171 µm day1 (mean 55 µm day1). There
was no significant heterogeneity of growth rate among chicks or weeks of
growth (P=0.475 and 0.093, respectively;
Table 2). However, the `long
bone' factor had a highly significant effect on growth rate
(P<105; Table
2), mainly because of growth rates 38% lower in the radius than in
other bones (P<0.002; multiple comparisons;
Table 2;
Fig. 4).
The `bone tissue type' factor had the strongest effect on growth rate (P<106; Table 2). Despite the four types having overlapping ranges (Fig. 4), laminar bone had significantly lower growth rates (P<103; multiple comparisons; Table 2), while radial bone had significantly higher growth rates (P<0.002; multiple comparisons; Table 2). Radial bone was the only tissue apposited at a rate above 109 µm day1. Longitudinal and reticular bone had similar intermediate growth rates. It is noteworthy that the effect of `bone tissue type' was measured while controlling for the `long bone' effect (and vice versa; principle of a two-way ANOVA). Moreover, a significant interaction between `long bone' and `bone tissue type' was detected (P<103; Table 2), which means that the two effects were not simply linearly additive. Graphical representation (Fig. 4) illustrates the results of the ANOVA; while the radius indeed has lower growth rates than other long bones, a shared pattern of increasing growth rate from laminar to radial bone is perceptible in all four long bones.
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Discussion |
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The range we report in king penguin chicks (7171 µm
day1) is congruent with those previous findings. Moreover,
it extends the known range of periosteal growth rates up to previously
unreported values; the highest rate was previously 112 µm
day1, observed locally in the Mallard chick
(de Margerie et al., 2002).
High-speed osteogenesis in early growth of the king penguin might be required
by peculiar life history traits in this species (i.e. large body size and only
3 months available to reach that size before winter starvation). Even the
Emperor Penguin (Aptenodytes forsteri), which is larger and hatches
further south on sea ice during austral winter, has a more evenly spread
growth, and thus may not grow as fast as the king penguin
(Stonehouse, 1960
; p. 60).
Test of Amprino's theory
According to our results, a relationship between growth rate and bone
tissue type does exist in king penguin chicks, even after controlling for
other factors (e.g. long bone); a special type of fibro-lamellar bone tissue
(i.e. radial bone) grows faster, while another type (i.e. laminar bone) has
comparatively moderate rates of growth. Nevertheless, aside from this central
result supporting Amprino's prediction, some other findings indicate that
Amprino's theory must be applied carefully.
First, we found a significant effect of `long bone' on growth rate, even
after controlling for the effect of `bone tissue type'. This means that a
given bone tissue type can have different absolute growth rates in different
parts of the skeleton. Starck and Chinsamy
(2002) also reported a `long
bone' effect on growth rate of longitudinal bone tissue in the Japanese quail.
Moreover, the significant interaction we observed between factors attests once
more that the relationship between bone tissue type and growth rate can vary
somewhat across parts of the skeleton.
Second, growth rate within each tissue type is highly variable, and ranges
extend widely. This point had been raised in other species
(Castanet et al., 2000;
de Margerie et al., 2002
;
Starck and Chinsamy, 2002
).
For instance, Starck and Chinsamy
(2002
) observed a range of
1050 µm day1 for longitudinal bone in the Japanese
quail. Consequently, when several tissue types are observed (as in the present
study), there is a consequential overlapping of growth rate ranges between
tissue types (Fig. 4), and some
differences fall below the significance threshold (e.g. comparison between
longitudinal and reticular bone; Table
2). Variations in porosity might explain such variation of growth
rates within tissue types (de Margerie et
al., 2002
; Starck and
Chinsamy, 2002
).
Far from nullifying Amprino's theory, which becomes more widely documented
after the present work, the preceding points still have restrictive outcomes.
A single and precise growth rate value can hardly be extracted from bone type.
Moreover, extrapolation of extant growth rates to fossils on the basis of a
shared bone tissue type (e.g. Curry,
1999; Horner et al.,
2000
; Padian et al.,
2001
) should be conducted very carefully, as already emphasised
(de Margerie et al., 2002
;
Starck and Chinsamy,
2002
).
Role of radial bone
Radial bone has the fastest growth and is the only tissue type found at
rates of 109171 µm day1
(Fig. 4). Radial bone has
already been observed in the bones of some wild or domesticated tetrapods
(Table 3), especially in
immature specimens, but its growth rate had never been measured. Nevertheless,
in the light of comparative paleohistological observations, de Ricqlès
(1977; p. 139) already
hypothesised that radial primary osteonal orientation could be the signature
of very rapid bone growth. Moreover, in aviculture, radial bone is observed in
the limb bones of birds artificially selected for rapid growth
(Dämmrich and Rodenhoff,
1970
; Itakura and Yamagiwa,
1970
; Leblanc et al.,
1986
; Leterrier and Nys,
1992
; Wyers et al.,
1993
). Finally, radial bone has not been observed in growth series
of mallard (Castanet et al.,
1996
; de Margerie et al.,
2002
), ostrich (Struthio camelus) or emu (Dromaius
novaehollandiae; Castanet et al.,
2000
), where growth rates did not attain the highest values
measured here in the king penguin. These results suggest that radial bone
could be a microstructural adaptation permitting higher rates of diametric
bone growth, within an already fast-growing tissue type (i.e. fibro-lamellar
bone). Functionally, the way through which the radial microarchitecture yields
faster growth remains unclear. This would require fine comparative
ultrastructural analysis of bone production by the periosteum, which was not
conducted here. Nevertheless, we do notice that radial struts of woven bone
can be produced continuously by the periosteum, contrary to a discontinuous
`saltatory' pattern in other bone tissue types (as illustrated in
Fig. 6).
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Biomechanical considerations
It is well-known that "mature function and growth are
antagonistic... features"
(Ricklefs et al., 1994). A
"trade-off between growth rate and functional maturity"
is a general constraint in various tissues of vertebrate organisms (review in
Ricklefs et al., 1998
). For
example, Carrier and Leon
(1990
) addressed a
"conflict between development and skeletal function".
Therefore, the question of the biomechanical outcome of very rapid bone growth
in the king penguin chick arises. Although this point would deserve
experimental investigations (i.e. mechanical testing), two facts already
support the idea that king penguin chick bone tissue has indeed poor
mechanical attributes: (1) high cortical thickness in chicks, as observed in
our material, is known to partly compensate for low resistance of the bone
tissue, as Carrier and Leon
(1990
) pointed out in the
California gull; (2) during their first month (i.e. before emancipation), king
penguin chicks have a parsimonious locomotor behaviour: a chick always remains
in the immediate vicinity of its parents (curled up in the brood patch or just
standing close to it). The needs for active locomotion are very low, as they
do not have to escape from predators, for example. As in other altricial
birds, growth itself, rather than body support and transmission of forces,
becomes the primary function of the bird's skeleton
(Starck, 1998
). Moreover,
research in aviculture has shown that birds artificially selected for high
growth rates tend to have weaker long bones (e.g.
Leterrier and Nys, 1992
),
sometimes resulting in pathological conditions
(Dämmrich and Rodenhoff,
1970
; Itakura and Yamagiwa,
1970
).
It is likely that radial bone has the most detrimental effect on mechanical
resistance, because radial cavities between bone struts interrupt the shear
flow around bone (Fig. 7) and
concentrate stresses at their corners. These `open section effects' are known
to dramatically reduce stiffness and strength, as has been modelled for
torsional loads (Elias et al.,
2000). Conversely, it has been proposed that laminar bone, which
incidentally grows more slowly, would have better mechanical properties
(de Margerie, 2002
).
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Adult king penguins exhibit strong
(Currey, 2002; p. 130), matured
bone (completed primary osteons, peripheral layer of lamellar-zonal bone,
intracortical Haversian bone, perimedullar layer of endosteal bone;
Meister, 1962
). The timing of
the bone maturation process in juveniles with regard to winter fast and to the
onset of full locomotor activity (i.e. departure to sea after the first year
on land) remains to be studied.
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Acknowledgments |
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