J Comp Physiol B (2000) 170: 253±260
Ó Springer-Verlag 2000
ORIGINAL PAPER
J. M. Durant á S. Massemin á C. Thouzeau
Y. Handrich
Body reserves and nutritional needs during laying preparation
in barn owls
Accepted: 25 February 2000
Abstract To investigate the composition of the body
reserves made during pre-laying by breeding European
barn owls (Tyto alba), we have analysed the body
composition of captive breeding and non-breeding females sacri®ced during the laying period. The data obtained were compared to the daily requirement for egg
formation obtained by an egg composition analysis and
the timing of egg synthesis. This study demonstrates that
body mass gain observed in breeding females (+38.3 g
after eggs in formation and gonadal tractus were removed) was not the consequence of an accumulation of
body fuels like lipids but of mainly water and lean material. The lipidic reserves were found to be less important in breeding than in non-breeding females and their
localisation modi®ed; lipids were absent from medullar
bones in breeding females which liberated room for
other storage. The subcutaneous tissue, which was
homogeneous in non-breeding females, was located
principally under the brood patch in breeding females.
Nutrients and energy required during egg formation
could be obtained without modi®cation of daily food
intake. These results show that a laying event can be
initiated in 14 days and that the onset of reproduction is
not triggered by body condition in barn owls. The water
accumulation observed is suggested to be the mere
consequence of an increase of protein metabolism (egg
and moult). These results intimate that the body mass
increase observed in diurnal and nocturnal raptors
during laying preparation, interpreted as an energy
safety strategy, ought to be reconsidered.
Key words Body reserves á Water á Egg synthesis
Bird of prey
Communicated by G. Heldmaier
J. M. Durant (&) á S. Massemin á C. Thouzeau á Y. Handrich
Centre d'EÂcologie et Physiologie EÂnergeÂtique,
Centre National de la Recherche Scienti®que,
67087 Strasbourg Cedex 02, France
e-mail: joel.durant@c-strasbourg.fr
Tel.: +33-38810/6917; Fax: +33-38810/6906
Abbreviations AAT abdominal adipose tissue á
BF breeding female á N-BF non-breeding
female á SAT subcutaneous adipose tissue
Introduction
The relative timing of an animal's acquisition and expenditure of resources is an important dimension of its
life-history. Some animals (``income breeders'') cover
reproductive expenditure by feeding during breeding,
whereas others (``capital breeders'') fuel reproductive
expenditure from foods eaten before laying which are
then stored prior to use (Drent and Daan 1980). This
dichotomy is developed to describe the energy acquisition strategy for breeding, and consequently it is less
obvious that it applies to the timing of speci®c nutrient
acquisition (Meijer and Drent 1999). For example, a
breeding bird could depend upon its body reserves for the
major part of its nutrient requirements but be entirely
dependent upon daily intake of one nutrient during laying. Such a micronutrient might be an essential amino
acid or calcium for egg-shell formation (Murphy 1994;
Perrins 1996; Houston 1998). Likewise, a bird typically
dependent upon daily food intake might need to store
certain nutrients over a long period before laying in order
to compensate for a peak demand that might not be met
through food consumption during laying. Consequently,
a bird can be a capital breeder for one nutrient and an
income breeder for another (Meijer and Drent 1999).
Raptors, both diurnal and nocturnal, are often classi®ed among income breeders because they strongly rely
on food intake during reproduction. Accordingly, their
clutch size is usually correlated with prey availability
(Newton and Marquiss 1981; Mikkola 1983; PietiaÈinen
et al. 1986; KorpimaÈki and Hakkarainen 1991; Wiebe
and Bortolotti 1995). However, the observation of a
di€erence between female body mass gain during prelaying and the mass of the clutch in formation in the
European kestrel (Falco tinnunculus) suggests that an
accumulation of body reserves may occur before the
254
laying period (Meijer et al. 1989; Meijer and Drent
1999). Alternatively, the body mass gain observed during the pre-laying period may re¯ect an increase of tissue
hydration rather than a nutrient reserve accumulation,
as has been recently observed in a passerine (Reynolds
and Waldron 1999). Moreover, body mass does not
decrease during laying in the European barn owl (Tyto
alba alba; Taylor 1994; Durant et al. 1996) which indicates that all the material stored during pre-laying was
not used for egg formation, even if the body mass gain
during pre-laying was correlated with the clutch size
(J.M. Durant unpublished data). In such a context, the
nature of this body mass gain in raptors and its role in
supplying nutrients or energy for egg formation needs to
be investigated.
The aim of this study is to examine the relationship
between female body mass gain and the requirements for
clutch formation in breeding barn owls. First we determined the composition of body reserves in breeding and
non-breeding captive female European barn owls. Secondly, we compared these reserves with the daily requirements for egg formation determined using data on
egg composition and the timing of egg synthesis.
Materials and methods
The study was performed on captive European barn owls from a
breeding programme conducted in the laboratory. Each pair of
owls was housed in an outside aviary (5 ´ 4 ´ 2.5 m) where a nest
box was installed. Owls were fed with living laboratory mice (Mus
musculus). During laying, females were fed exclusively by males
who brought prey to the nest. Laying occurred between March and
October, the average temperature ranging between 10 °C and
28 °C. Females were regularly weighed to the nearest 0.1 g.
Female body composition
The body composition of ®ve breeding females (BFs) during laying
was compared to that of ®ve non-breeding females (N-BFs) during
the reproduction period. BFs were sacri®ced after 3.5 days without
laying, a period we assumed indicated the end of the laying event (a
little over 1 day after the average laying interval of 2.3 days for
barn owls; J.M. Durant, unpublished data). N-BFs were selected
on body mass criterion (steady body mass) and their status con®rmed by the ovary analysis. Euthanasia was performed under
halothane anaesthesia (Close et al. 1997).
Dissection was conducted stepwise (Massemin et al. 1997;
Thouzeau et al. 1997). Skin, adipose tissues, gonadal tissues (oviduct and ovary), liver, right pectoral, right humerus and right femur were removed from the body and weighed (fresh mass).
During the dissection some water was lost. Repeated weighing at
each step of the dissection of the separated tissues allowed an accurate correction for this water loss. Each tissue was freeze-dried
and reweighed (dry mass). The water content corresponds to the
di€erence between fresh mass and dry mass. Each tissue was
ground under liquid nitrogen into a ®ne homogeneous powder
(3 mm mesh) and aliquots taken for assays.
Nitrogen content, from a 100-mg aliquot was determined by the
Kjeldhal method and converted to protein by multiplying by 6.25
(Schmidt-Nielsen 1997). Total lipid content was measured gravimetrically by a method adapted from Folch et al. (1957) on 1-g
aliquots of powder. Lipid extraction was repeated twice for each
tissue. Mineral content was determined after total combustion of a
1-g aliquot in a furnace at 800 °C for 24 h.
Egg composition
Of 15 freshly laid eggs from 6 di€erent broods, the whole egg as
well as the separated yolk and shell (including attached membranes) were weighed to the nearest 0.01 g. Albumen mass was
estimated by subtracting the sum of the masses of yolk and shell
and membranes from the whole egg mass. The components were
freeze-dried to constant mass (dry mass) and water content was
calculated. Yolk and albumen were then ground into a ®ne and
homogeneous powder and aliquots were taken for analysis.
Nitrogen, lipid, mineral and water contents were determined in
a way similar to the analysis of body composition. Protein content
was calculated by multiplying nitrogen by 6.4 for the yolk and 6.7
for the albumen (calculated from average nitrogen content of yolk
and albumen proteins; Romano€ and Romano€ 1949). Energy
content was taken as 16.7 kJ g)1 for the protein component and
37.7 kJ g)1 for the lipids (Grau 1996).
In¯uence of the date of sacri®ce of BFs
Even though the ®ve BFs were sacri®ced after 3.5 days without
laying, not all the females had laid their last egg. In two cases, an
egg was found inside the oviduct, its status as last egg con®rmed by
the presence of atretic follicles inside the ovary. Therefore, the
euthanasia was performed at di€erent stages within the laying period, from 2.3 days before to 3.5 days after laying of the last egg.
This heterogeneity was due to the female's faculty to postpone
laying which increases the time interval before the last laying. We
tested the in¯uence of this heterogeneity of sacri®cial date on body
composition. No relationship was found between the body composition (expressed as mass or content, fresh or dry, for water,
lipid, protein or mineral) and the sacri®cial date during laying
(expressed as percentage of the total time necessary for laying
completion) nor with the size of the clutch (regression analysis,
P > 0.05).
Body mass loss during laying (6.9 ‹ 0.7 g) represented less
than 2% of the body mass before the ®rst laying, if we did not take
into account body mass variation due to egg formation and oviposition. The daily body mass loss throughout the laying events
was low, with a mean value of 0.50 ‹ 0.07 g day)1. Accordingly,
laying can be considered a period of steady body mass.
Material rate deposition and daily requirements
The daily deposition of egg material in the reproductive organs was
obtained from data on egg composition and egg deposition. To
make this calculation, it was necessary to take into account the
developing yolks resorbed before oviposition, i.e. the atretic follicles. The timing and rate of yolk deposition used to make the calculation are as follows (J.M. Durant, unpublished data): the delay
between two successive egg synthesis initiations is taken to be 2.3
days, and egg deposition took on average 14 days from initiation to
laying. The yolk deposition rate follows a second-order equation
(Yolk mass ˆ )0.1279 + 0.2543 ´ day + 0.0128 ´ day2; range of
validity from 1 to 12 days) and is ®nished 2.4 days before laying.
Albumen, membranes and shell are assumed to be deposited during
this period, i.e. after ovulation, in the same place along the oviduct
and with the same rate as in other birds (Taylor 1970; Grau 1984).
The number of yolks deposited is greater by two than the number of
eggs laid. The two surplus yolks are lost through atresia at a rate of
20% per day after the ovulation of the last egg of the clutch.
The egg material deposited in the reproductive organs on a given
day is the sum of the eggs in formation at this stage independently of
their fate (laying or atresia). The average daily deposition of egg
material is the sum of the total deposition divided by number of
days necessary for clutch completion (30 days). The calculation was
made for each nutrient. The average daily requirement for a given
nutrient is the daily deposition to which was applied its assimilation
eciency (Table 1). The assimilation eciency in energy or nutrient
was calculated using the following formula:
255
Table 1 Mouse composition and assimilation eciency by barn
owls. (GI gross intake, which was determined by subtracting food
scraps from to the food supply, PA part assimilated, which corresponds to GI, pellet content ± excreta content)
Water
Lipid
Protein
Mineral
Energy
a
b
Fresh mass content
(%) (n = 6)
Assimilation eciency
(%) = PA/GI ´ 100
71.3 ‹
7.3 ‹
18.2 ‹
3.2 ‹
(6.6 ‹
100
90a
59.4b
21.5b
72.3b
1.0
0.4
0.9
0.1
0.2 kJ g)1)
Place 1996
J.M. Durant, unpublished data
N-BFs. BFs had signi®cantly more water, proteins and
minerals (by 44.6 g, 6.2 g and 2.9 g respectively) but less
lipid (15.4 g) than N-BFs. The increase in body water
was not only due to a gain in protein since the water/
protein ratio was also signi®cantly higher. When expressed as fresh mass content, the two groups di€ered
only in water (11% higher in BF) and lipid content (44%
lower in BF). When expressed as dry mass content, the
two groups di€ered in lipids (33% lower in BF), proteins
(18% higher in BF) and mineral content (26% higher in
BF).
Tissue composition
gross intake ± pellet content ± excreta content=gross intake 100
where the growth intake corresponds to the amount of ingested
energy or nutrient calculated by subtracting the content of food
scraps from the content of the food provided. The energy cost for
biosynthesis was taken as 0.82 kJ kJ)1 for protein and 0.28 kJ kJ)1
for the lipid (Weathers 1996). The requirement in number of mice
consumed was calculated using the average body composition of
laboratory mice, determined in a way similar to that of the owls'
body composition.
Statistical analysis
Data presented in the tables and text are means ‹SE. Comparisons
between data were performed using the Mann-Whitney U-test. The
Friedman analysis of variance by ranks test was used to compare
repeated measurements on the same subjects.
Results
Female body composition
The BFs were signi®cantly heavier in total body mass
(by 52 g, Table 2) than the N-BFs. The fresh body mass
of BFs (total body mass minus feathers minus gonadal
tissue) was signi®cantly heavier (by 38.3 g) than that of
Table 2 Whole body composition of N-BF and BF barn owls.
[Fbm Fresh body mass (total
body mass without feathers and
gonadal tissue), ns not signi®cant, Tbm total body mass
(body mass without pellet, digestive content and egg in the
oviduct)]
The whole muscular mass was estimated by calculation
for BFs and N-BFs (Table 3). With the whole skin (skin,
subcutaneous adipose tissue -SAT- and adipose tissue
deposited on muscles), it accounted for 70% of the total
body mass gain in BF and for 95% of the fresh body
mass gain. Muscles contributed for 62% of the fresh
body mass di€erence between females, and pectorals
alone for 16%. The body water increase is explained by
a higher water content (%) in di€erent organs of BFs
(e.g. pectorals and liver). Fresh mass and water content
(%) of the whole skin without lipids were signi®cantly
higher in BFs.
In addition to the di€erence in lipid composition (in
mass, fresh and dry content) found for the whole body,
di€erences in lipid allocation appeared in fatty tissues
between the two groups of females. Two fatty tissues
showed important di€erences in lipid allocation: the
whole skin and the bones. Despite the di€erence of
whole skin fresh mass, the lipid mass was not di€erent
between BFs and N-BFs. During dissection, the homogeneity of the SAT in N-BFs made its separation from
the skin dicult. On the contrary, in BFs, SAT was
easily isolated because it was principally located under
N-BF (n = 5)
BF (n = 5)
311.6
277.2
276.7
164.8
111.9
41.6
55.4
14.9
3.0
5.0
6.5
6.3
2.0
4.4
3.3
0.8
0.4
0.1
363.1
329.1
315.0
209.4
105.6
26.2
61.6
17.8
3.4
‹
‹
‹
‹
‹
‹
‹
‹
‹
5.5
4.9
3.7
3.7
3.3
3.2
0.6
0.8
0.1
0
0
0
0
6
2
0
0
0
0.008
0.008
0.008
0.008
ns
0.032
0.008
0.008
0.008
Fresh mass content in percentage of Fbm
Water
59.7 ‹ 0.7
Lipid
14.9 ‹ 0.9
Protein
20.0 ‹ 0.2
Mineral
5.4 ‹ 0.1
66.5
8.3
19.6
5.6
‹
‹
‹
‹
1.0
1.0
0.2
0.2
0
0
4
12
0.008
0.008
ns
ns
Dry mass content in percentage of Dbm
Lipid
36.8 ‹ 1.5
Protein
49.8 ‹ 1.3
Mineral
13.4 ‹ 0.4
24.5 ‹ 2.3
58.6 ‹ 2.0
16.9 ‹ 0.6
0
0
0.008
0.008
0.016
Mass in grams
Total body mass (Tbm)
Tbm ± feathers
Fresh body mass (Fbm)
Body water
Dry body mass (Dbm)
Body lipid
Body protein
Body mineral
Water/protein
‹
‹
‹
‹
‹
‹
‹
‹
‹
U-test
P-value
256
Table 3 Tissue mass and composition of N-BF and BF barn
owls. [AAT Abdominal adipose
tissue including all adipose tissues in the thoracic and abdominal cavities, Gonadal tissue
oviduct and ovary (containing
the eggs in formation), Muscles
whole muscles of the body estimated by calculation, ns not
signi®cant, WS whole skin
(skin, subcutaneous adipose
tissue and adipose tissue deposited on muscles)]
N-BF (n = 5)
BF (n = 5)
Fresh mass in grams
Gonadal tissue
Muscles
Pectoral (right)
Liver
Whole skin (WS)
WS without lipids
0.57
136.9
17.3
7.1
31.0
18.9
‹
‹
‹
‹
‹
‹
0.25
2.1
0.5
0.3
2.8
1.5
14.11
160.4
20.4
10.6
43.3
28.8
‹
‹
‹
‹
‹
‹
2.13
1.8
0.3
0.3
1.8
1.0
0
0
2
0
0
0
0.008
0.008
0.032
0.008
0.008
0.008
Dry mass in grams
Humerus
Pectoral (right)
Liver
Whole skin
WS without lipids
1.28
5.5
2.2
18.1
6.0
‹
‹
‹
‹
‹
0.03
0.3
0.1
2.2
0.2
1.22
5.4
2.9
20.9
6.5
‹
‹
‹
‹
‹
0.03
0.1
0.1
2.0
0.2
5
12
0
7
5
ns
ns
0.008
ns
ns
Water content in %
Pectoral (right)
Liver
Whole skin
WS without lipids
68.1
69.4
42.2
68.0
‹
‹
‹
‹
1.2
0.4
2.8
1.1
73.7
72.9
52.1
77.5
‹
‹
‹
‹
0.2
0.5
3.4
0.8
0
0
3
0
0.008
0.008
ns
0.008
Lipid
AAT (in grams)
Whole skin (in grams)
Femur (in % of dry mass)
4.8 ‹ 1.1
14.8 ‹ 1.9
23.9 ‹ 3.7
1.8 ‹ 0.8
11.8 ‹ 2.0
2.5 ‹ 0.2
3
9
0
ns
ns
0.008
Mineral
Humerus
Femur
61.4a
41.0a
66.6 ‹ 0.1
58.2 ‹ 1.7
a
U-test
P-value
Not measured here (Thouzeau et al. 1997)
the brood patch, and contained 70 ‹ 1% of the lipids of
the whole skin. Brood patches were signi®cantly larger
in BFs (55.8 ‹ 6.0 cm2 vs. 12.8 ‹ 0.3 cm2 in N-BF;
U-test, P < 0.001).
Lipids of bone were nearly exhausted in BFs with
only 10% of the content of N-BF bones left.
Egg composition
An average barn owl's egg weighing 18 g comprises 77%
water, 9% proteins, 8% minerals, and 6% lipids, which
corresponds to a total energy content of 65 kJ (Table 4).
Seventy-six percent of the total water is contained in the
albumen. Proteins are stored predominantly in the albumen and the shell membranes (70% of egg proteins).
Albumen alone contained 59% of the proteins. Lipids
were stored principally inside the yolk (Table 4) and
represented 65% of its dry mass. Over 96% of the egg's
minerals were stored in the albumen and predominantly
in the shell (91%). Due its high lipid content, the yolk
contained 71% of the energy content.
Material deposited for the formation of a clutch
of six eggs
During the formation of a six-egg clutch, the maximum
mass of egg material present in the gonadal tissue at the
same time was around 30 g. This mass represents only
1.7-times the mass of one egg (Fig. 1) whereas six eggs
were developing. The maximum egg material deposition
occurred the day of the ®rst egg laying. Seventy-two
percent of this mass gain was water (21.0 ‹ 0.3 g). The
remainder of the gain included proteins for 10%
(2.98 ‹ 0.04 g), lipids for 13% (3.67 ‹ 0.06 g) and
minerals for 5% (1.33 ‹ 0.03 g).
The daily egg formation and its requirements (average and maximum) for each nutrient were calculated for
the production of a six-egg clutch (Table 5). The most
deposited nutrient each day by mass was the water. The
maximum daily need for water (8.36 ‹ 0.11 g day)1)
Table 4 Composition and energy content of barn owl eggs
(n = 15). Shell measurements include shell membranes
Total
Yolk
Albumen
and shell
Mass in grams
Fresh mass
Dry mass
Protein
Lipid
Mineral
17.6
4.10
1.62
1.00
1.48
Energy in kJ
Protein
Lipid
27.1 ‹ 0.5
37.8 ‹ 0.6
8.3 ‹ 0.2
37.5 ‹ 0.6
18.8 ‹ 0.5
0.3 ‹ 0.1
64.9 ‹ 0.8
45.8 ‹ 0.7
19.1 ‹ 0.5
29.8 ‹ 0.1
10.8 ‹ 0.2
7.5 ‹ 0.1
1.4 ‹ 0.1
Total
‹
‹
‹
‹
‹
0.2
0.06
0.03
0.02
0.04
4.3
1.54
0.49
1.00
0.05
‹
‹
‹
‹
‹
0.1
0.02
0.01
0.02
0.01
13.3
2.56
1.13
0.00
1.43
‹
‹
‹
‹
‹
0.3
0.06
0.03
0.00
0.04
)1
Energy content in kJ g
Dry mass
15.8 ‹ 0.2
Fresh mass
3.7 ‹ 0.1
257
Discussion
Changes in the body composition of female barn owls
were investigated during the egg-laying period. This
study contrasts with the generally accepted hypothesis
that some diurnal and nocturnal raptors accumulate
energy reserves before laying (Meijer and Drent 1999).
The body mass gain observed in breeding barn owls was
not the consequence of an accumulation of lipids (energy
reserves) but principally of water, as observed in passerines (Reynolds and Waldron 1999), and also proteins.
Furthermore, the lipid stores were even lower in breeding than in non-breeding barn owls and the localisation
of these stores was modi®ed.
Fig. 1 Nutrients deposited in the female reproductive organs for a
total clutch of six eggs with a laying interval of 2.3 days. The dashed
line represents the nutrients deposited for a single egg. Each peak
corresponds to the completion and the laying of one egg. The number
over each peak corresponds to the number of eggs in formation at that
time. Albumen, shell membranes and shell are deposited the last 2
days inside the oviduct after ovulation. The decrease in nutrient
deposition after clutch completion corresponds to the resorption of
two follicles that had begun their rapid yolk deposition
and minerals (1.25 ‹ 0.03 g day)1) occurred 1 day
before the laying of the ®rst egg, during albumen
plumping and shell deposition respectively; the two egg
components contained the highest percentage of water
and minerals. The maximum daily requirement of protein (1.05 ‹ 0.02 g day)1) occurred before the laying of
the ®rst egg during the lag interval, i.e. during the albumen dry matter deposition. The maximum daily lipid
requirement (0.43 ‹ 0.01 g day)1) occurred 3 days before laying the ®rst egg when the quantity of yolk material deposited in the ovary was highest. In mice per
day, the most demanding nutrient was the minerals, with
a maximum requirement of 7.2 mice day)1.
Table 5 Average daily requirements for the formation of a clutch
of six eggs. Daily egg formation and its requirements were calculated by dividing the total requirements by the number of days
necessary for the clutch completion (30 days). The cost for synthesis of lipid and protein was calculated using eciency values found
in Weathers (1996). The energy requirement was calculated using
the assimilation eciency and the energy content (see Table 1) for
each nutrient. Daily food requirements in energy and in number of
Body composition
Water gain during laying preparation
Not considering mass gain through hypertrophy of reproductive tissues, eggs in formation and mass variation
due to the gastric and intestinal contents, BFs were still
around 40 g heavier than N-BFs, i.e. an increase of 14%
of body mass. Gonadal tissue will not be considered in
the following discussion as its mass was still high (25
times higher) at the end of laying compared to gonadal
tissue of non-reproductive owls (Table 3).
The major part of the body mass gain observed was
due to an overall hydration of tissues, such as muscles,
liver or skin. Water accumulation, particularly in muscles, may be a consequence of changes in metabolism for
reproduction (Reynolds and Waldron 1999). Indeed,
protein metabolism (synthesis and catabolism) is often
associated with a modi®cation of water balance. A water
accumulation has been shown prior to an enhanced
protein synthesis as occurs during moulting (Cherel
et al. 1994) and growth (Ricklefs 1983). This could
probably explain the high hydration factor of the skin of
mice consumed are calculated using the average body composition
of a laboratory mouse weighing 25 g. Date is the number of days
before the ®rst egg is laid when the period of maximum requirement began. The requirement in mice, for proteins and lipids, was
calculated using the total requirement during this period divided by
the duration. For water and minerals the requirements are the total
for the period considered
Daily egg formation in
Daily food requirement in
Period of maximum requirement
Mass
(g day)1)
Energy
(kJ day)1)
Mice
per day
Date
(day)
Mice number
day)1
)0.7
)2.3
)3.8
)0.5
0.3
0.5
0.4
7.2
‹
‹
‹
‹
Energy
(kJ day)1)
Water
Protein
Lipid
Mineral
2.70
0.32
0.21
0.27
0.04
0.01
0.01
0.01
0
7.6 ‹ 0.1
8.5 ‹ 0.1
0
0
12.8 ‹ 0.2
9.4 ‹ 0.1
0
0.2
0.1
0.1
1.5
Total
Synthesis
3.50 ‹ 0.05
16.1 ‹ 0.2
8.6 ‹ 0.1
22.2 ‹ 0.4
12.8 ‹ 0.1
0.1
0.2
258
breeding barn owls since in this species moulting for
females takes place during incubation (Taylor 1994).
Likewise, during protein catabolism, such as the protein
breakdown at the end of a prolonged fast (Thouzeau
et al. 1999), there is a liberation of preformed water.
While there was not an important change in protein
mass, we cannot discard the possibility that there may be
a substitution of certain proteins for others. The metabolic consequences of such protein substitutions could
be water accumulation, e.g. to counterbalance a change
in osmotic equilibrium due to amino acid ¯ux. During
egg formation, the requirement for speci®c amino acids
may be high in spite of their scarcity in the diet (Murphy
1994; Houston 1998). These requirements might imply
that female barn owls use speci®c amino acids stored
beforehand as proteins, e.g. in muscles such as the pectorals (review in Houston 1998). In addition to water, a
moult is very demanding in speci®c amino acids like the
cystine (Murphy and King 1991; Dietz et al. 1992;
Cherel et al. 1994) stored most notably in liver proteins.
The release of stored cystine and the associated preformed water could explain the high hydration of a BF's
liver. Moulting and egg synthesis are two demanding
processes for water and proteins and thus resources must
be split between them. This might imply that BFs need
water and protein reserves to conclude successfully both
processes simultaneously.
Relocation of lipids during laying preparation
There was no accumulation of body lipid in BFs, as
would be expected if energy reserves are accumulated as
has been proposed (Wijnandts 1984; Hirons 1985; Taylor 1994). On the contrary, there was less body lipid in
BFs than in N-BFs. This decrease indicates a utilisation
of body lipids during pre-laying. One explanation could
lie in a higher lipid requirement, i.e. energy requirement,
in a BF than in an N-BF. However, this hypothesis
seems unlikely, since, when fed ad libitum in our aviaries, the food intake of BFs did not change signi®cantly
over the periods studied (3.8 ‹ 0.2 mice day)1; Friedman test, v22 ˆ 2.235, n ˆ 13, P ˆ 0.389). Moreover, the
decrease of body lipid (more than 15 g) in the BF was
greater than the requirement necessary to form a clutch
of six eggs (approximately 6 g). Consequently, the lipid
requirement for egg formation does not explain all the
lipid utilisation observed. On the other hand, lipids were
nearly exhausted in BF bones. Such a low lipid content
has been observed only in bones of barn owls after a
prolonged fast (Thouzeau et al. 1997), where it was
demonstrated that in contrast to other fat deposits, bone
lipids were only mobilised during the last stage of the
fast, when a shift from lipid to protein fuel metabolism
occurred. Obviously, the low lipid content of the
medullar bones in BFs is not due to such a depletion of
body reserves. One explanation would be that the decrease of bone lipid makes possible a higher storage of
other nutrients, such as minerals and water, without
modi®cation of bone volume. Female birds are well
known to store minerals in their bones during laying
(Clunies et al. 1993; Perrins 1996; Houston 1998). Accordingly, the femur and humerus of breeding barn owls
had higher mineral contents than those of non-breeding
owls (Table 3). Likewise, a decrease of the abdominal
adipose tissue (AAT) could be necessary to accommodate the growth of the reproductive tissue inside the
abdomen.
Another explanation for the body lipid decrease in
BFs could be that part of the lipids disappear in the
process of being displaced in the body. This hypothesis is
suggested by the di€erence of lipid localisation in the
skin of BFs compared with N-BFs. Despite the same
lipid mass contained by the whole skin, the SAT of the
brood patch was thicker in BFs. This may be due to a
migration of lipids to brood patch SAT from other depot fat stores such as other part of the SAT. This
movement was related to an increase of the size of the
brood patch for reproduction, probably before laying.
The accumulation of adipose tissue under the brood
patch, which is a de-feathered surface, could be an adaptation to limit heat loss and/or adjust heat transfer to
the clutch. The heat transfer necessary to warm the eggs
would then be maintained by the peripheral blood ¯ow
in the brood patch (MidtgaÊrd et al. 1985).
Requirements for clutch synthesis
The yolk and water contents of barn owl eggs are typical
for an altricial species (Carey et al. 1980). Relative to
two raptors of similar size, one diurnal and one nocturnal, the European kestrel (5.1 kJ g)1 fresh egg mass,
Meijer et al. 1989) and the long-eared owl (Asio otus;
4.4 kJ g)1, Wijnandts 1984), the energy content of barn
owl eggs (3.7 kJ g)1) is low. The female barn owl can
thus lay larger clutches for the same total energy cost.
Furthermore, for the same clutch size the daily energy
expenditure would be lower than in the other two species. In comparison with the average value for altricial
birds (4.4 ‹ 0.1 kJ g)1; calculated from Vleck and
Vleck 1996) the energy content of barn owl eggs is still
low.
Considering a delay between two successive egg initiations of 2.3 days, the development of a seventh egg is
calculated to begin 0.8 days after the laying of the ®rst
egg of the clutch, which occurred after 14 days of development. Based on this timing and independently of
clutch size, there would never be more than six eggs
developing at the same time inside a barn owl ovary. For
a clutch of six eggs with two atretic follicles (Fig. 1), the
period of high nutrient requirements occurs from 1.5
days before the laying of the ®rst egg to the third laying,
i.e. in the period when six eggs are developing at the
same time. During this period, the nutrient requirements
are highest prior to each laying when the deposition rate
is maximum, i.e. equivalent to the daily maximum requirements calculated before the laying of the ®rst egg.
259
Despite the lower cost of clutch synthesis, it may be
that one or more of the resources necessary to form an
egg (water, protein, lipid, mineral) constitutes a limiting
factor during whole or part of the egg formation. By
mass, the predominant nutrient for egg synthesis is
water. However, water is easily obtained since it constitutes the major part of the prey and is easily assimilated. Conversely, eggs contain relatively few minerals
(8.4% of fresh mass), which constitute only a small part
of the prey and are not well assimilated (22% eciency,
Table 1). The daily mice requirement to provide
sucient minerals for egg formation is nine times greater
than for any other nutrient (Table 5). The time of the
maximum mineral requirement, which theoretically
requires the consumption of seven mice day)1, is during
the day preceding oviposition, which is the period of
shell deposition, whereas the females ate 3.9 ‹ 0.4
mice day)1 at this time. During the ®nal 15 h of shell
formation, the calcium secretion rate is so high that
circulating blood calcium would be depleted in a few
minutes if not continually replenished by increased
intestinal absorption and bone mobilisation (Clunies
et al. 1993). The fact that food intake did not change
during reproduction suggests that barn owls set aside
minerals for shell deposition before each laying. Some
tissue may thus act as a temporary storage pool for
calcium over the egg laying cycle (Houston 1998). This
storage could be located in medullary bones which
would then serve as a labile source of calcium for
formation of the egg shell (Reynolds 1997) as suggested
by the results obtained for the bone mineral content.
Since eggs are laid with an interval of at least 2 days, the
food intake between layings would suce to ful®l the
requirements for shell formation if calcium is stored.
The majority of the nutrients required for egg formation can be obtained by routine food intake; only
minerals need to be temporarily accumulated in bones
during pre-laying. As a consequence, female barn owls
do not need much time to begin a new laying attempt.
As observed in our aviaries, a second clutch can be laid
14 days after the ®rst clutch. Moreover, females were
observed to lay a clutch only 4 weeks after having been
subjected to a prolonged period of total food deprivation up to a relative body mass loss of 30% (Handrich
et al. 1993). This implies that the climatic conditions
during winter and the resulting poor body condition do
not have a long-term in¯uence on reproduction. Reproduction does not depend on stored energy and materials and is thus not triggered by reaching an optimum
body condition. The fact that the gain in body mass
observed during the pre-laying period is not the consequence of the accumulation of nutrient reserves but of
water reinforces this conclusion.
To conclude, the gain in body mass observed in
breeding barn owls is not due to an accumulation of
energy reserves but of an accumulation of water. The
nutrients required for egg formation can be obtained by
routine food intake during egg formation. These results
indicate that barn owls fall nearer the ``income'' end of
the capital-income continuum. The water accumulation
observed in breeding barn owls con¯icts with the hypothesis that nocturnal female raptors make energetic
reserves as a bu€er against food scarcity during incubation (Taylor 1994; Wijnandts 1984; Hirons 1985).
Despite the small number of owls analysed, this study
highlights the importance of water accumulation in
avian reproduction.
Acknowledgements We thank Alexandre Garnier for help with the
laboratory analyses. We also thank Jim Reynolds for useful comments on an early draft of the manuscript and Fabienne Alma,
Guillaume Froget and Arthur Pape for editing aid. The experiments were done in compliance with current French laws and after
program acceptance by French authorities: Authorisation of the
MinisteÁre de l'Agriculture et de la PeÃche no04196.
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