Post-hatching parental care behaviour and hormonal status in a
Boos, Mathieu1,2, ZIMMER Cédric1, Carriere Aurélie1, Robin Jean-Patrice1, and Odile
1 Institut Pluridisciplinaire Hubert Curien, Département Ecologie, Physiologie et
Ethologie, UMR 7178 CNRS-ULP, 23 rue Becquerel, 67087 Strasbourg Cedex 2,
France. Phone: +33 3 88 10 69 24; Fax: +33 3 88 10 69 06
2 Naturaconst@ 14 rue principale F-67 270 Wilshausen.
Phone / Fax: +33 3 88 02 26 76.
Correspondence: Odile PETIT, firstname.lastname@example.org
In birds, the link between parental care behaviour and prolactin release during
incubation persists after hatching in altricial birds, but has never been precisely studied
during the whole rearing period in precocial species, such as ducks. The present study
aims to understand how changes in parental care after hatching are related to circulating
prolactin levels in mallard hens rearing ducklings. Blood was sampled in hens over at
least 13 post-hatching weeks and the behaviour of the hens and the ducklings was
recorded daily until fledging. Contacts between hens and the ducklings, leadership of
the ducklings and gathering of them steadily decreased over post-hatching time.
Conversely, resting, preening and agonistic behaviour of hens towards ducklings
increased. Plasma prolactin concentrations remained at high levels after hatching and
then fell after week 6 when body mass and structural size of the young were close to
those of the hen. Parental care behaviour declined linearly with brood age, showed a
disruption of the hen-brood bond at week 6 post-hatching and was related to prolactin
concentration according to a sigmoid function. Our results suggest that a definite
threshold in circulating prolactin is necessary to promote and/or to maintain
post-hatching parental care in ducks.
Keywords: prolactin, hen-brood bond, post-hatching period, Anatidae.
In birds, parental care behaviour is a key concept of parental investment.
According to life-history traits, it is selected for differently among bird species and
relies on the trade-off between increasing survival of the offspring while impairing that
of the parents for future reproduction (Kear, 1970; Afton and Paulus, 1992). Trivers
(1974) has suggested that the parent-offspring conflict is expected to increase during the
period of parental care, and offspring are expected to compete with their parents. During
incubation, parental care is linked to prolactin secretion in a large number of species
(Schradin and Anzenberger, 1999). However, after hatching, although it has been
shown that this hormone is involved in the control of parental behaviour in altricial birds
(Silverin and Goldsmith, 1984; Hall et al., 1986; Schoech et al., 1996; Lormée et al.,
1999), its role in precocial species is more controversial (Goldsmith, 1991). Indeed,
depending on the species, either sharp drops or slow decreases of adult prolactin levels
after hatching have been reported (Goldsmith and Williams, 1980; Dittami, 1981; Oring
et al., 1986; Hall, 1987; Oring et al., 1988). While data for common eiders, Somateria
mollissima, suggest that a threshold prolactin level should be reached to promote
parental care (Criscuolo et al., 2002), the direct link between the hormonal status and
the intensity of this behaviour has in fact never been specifically explored.
To investigate such a possible hormonal-behavioural relationship in precocial
birds, the mallard duck Anas platyrhynchos is a suitable model. In this species, a drop in
prolactin plasma concentration has been reported just after hatching (Goldsmith and
Williams, 1980; Hall, 1987), while a strong hen-brood attentiveness has been observed
at least during the first 2-3 weeks post-hatching, before a complete disruption of the
bond in about 6-7 weeks old ducklings (i.e. 2-3 weeks before fledging, Talent et al.,
1983; Afton and Paulus, 1992). From these data it could be concluded that if prolactin is
still involved in parental care after hatching in such species, plasma levels of prolactin
should be correlated with the intensity of the hen-brood bond and be maintained above a
threshold value. To check this hypothesis, we performed here the first detailed study of
parental care behaviours in mallard females during the rearing of ducklings, from
hatching to fledging, in relation to the hormonal status of the females over the same
period. To take into account possible effects of the photoperiod on prolactin secretion
and nest abandonment (Hector and Goldsmith, 1985; Bluhm, 1992; Lormée et al., 1999;
Sockman et al., 2004) the present study was undertaken over spring and summer.
Together with prolactin, plasma levels of corticosterone, known to interact with
prolactin release (Criscuolo, 2001; for review see Chastel et al. 2005) and testosterone
(potentially involved in aggressive behaviour, Dittami, 1981) were also measured.
MATERIALS AND METHODS
Animals and experimental condition.
The present study was conducted in 2003 on 7 adult mallard pairs obtained from
the registered breeding field station of the Canarderie de la Ronde (Cère la Ronde,
France). About 2 months before the nesting period, the pairs were held in separated
outdoor pens (12 m²), each including a nest and a 1m² basin provided with clear running
water. Each side of the pen (except the top) was opaque to avoid any visual contact
between the pens and to prevent any external stress and especially from the observers
during periods of behaviour monitoring. After laying and as soon as the hen started
incubation, males were removed from the pen. Ducklings hatched between 15 April and
23 June and were continuously held with the hen until mid September, i.e. well after
The ducks were subjected throughout the study to ambient temperature and
natural photoperiods. A balanced commercial food adapted for adults and one specific
for ducklings (Sanders Corporation), along with fresh drinking water, were provided ad
Blood sampling and weighing
Blood samples were taken from all hens every 3/4 days during at least 13 weeks
from hatching, between 10:00 and 12:00 am. Hens were caught in the pen with a net and
transported to a nearby room. Within three minutes after capture, blood was collected
into a heparinized tube by puncture of the brachial vein, centrifugated and then plasma
was frozen at -20°C until analysis. Before being released back to the pen, the hen was
weighed (±1g) and the post-nuptial moulting status of the plumage was determined by
visual examination. Three scores were distinguished: no moult, body moult (feathers
along side and flanks), moult of wing feathers. Body mass was used as a condition index.
No size corrections were applied since structural measurements do not significantly
explain body reserves in hen mallards and can be misleading (Boos et al., 2000; Green,
2001). Hens were not separated from the brood for more than 10 minutes. Once a week,
ducklings were weighed (± 1g) and immediately released in the pen. Ducklings were
caught as groups of 3 or less so that the female was always in contact with some of her
Hormonal plasma levels were determined by radio-immunoassay. Prolactin
concentration was determined at CEBC in Chizé (France) using an avian antibody for
prolactin (see Lormée et al., 1999, 2000). Testosterone and corticosterone assays were
made in our laboratory using 125I RIA kit double antibody from ICN Biomedicals
(http:/www.icnbiomed.com). All plasma samples were assayed at the same time in
duplicate to eliminate inter-assay variation. The intra-assay coefficient of variation was
5.5% (n=6 duplicates) and 2.0% (n=8 duplicates) for prolactin and corticosterone
respectively (values could not be determined for testosterone because of detection level,
see results section).
Females and ducklings were observed according to the focal sampling method
(Altmann, 1974) five times a week by the same observer (A. C.). On the days blood was
sampled, no other observations except the recording of vocalization emitted by the hen
at release, were performed. Each focal observation lasted 40 min and was carried out
between 15:50 and 19:30. From preliminary observations conducted in the field one
year before, this period corresponded to maximum daytime activity (Carrière,
unpublished data). Observations were randomized weekly so that a subject observed the
first day at 15:50 was observed at, for example, 18:50 the following day. Observations
started one day after hatching, i.e. when ducklings had left the nest (Kear, 1970).
Behavioural monitoring was carried out over 9 weeks, until ducklings fledged (Gollop
and Marshall, 1954).
The observer was outside of the pen in an opaque black cabin so that she could
not be seen by the ducks. The different behaviours displayed by the animals were
monitored through a Plexiglas plate covered with two-way mirror film. The time and
duration of each behaviour displayed by the ducklings and the female were recorded
with a stopwatch (± 1s).
One year before the present study, a specific ethogram was established in the
field on an independent group of free-ranging mallards. Behaviours were classified into
four principal categories:
Self-maintenance behaviours of the female:
- Foraging: taking food items both in water and on the ground.
- Locomotion: walking and flying.
- Swimming: locomotion in water.
- Preening: cleaning the plumage with the bill or immersion of the head and
the neck followed by a rapid raise of the body to sprinkle the back.
- Resting: head placed on the back or the bill under the scapulars.
Intensity of the hen-brood bond:
Close proximity i.e. body contacts and distance (<1 m) between the hen
and the offspring (duration in s).
Hen being distant (> 1 m) from the ducklings (duration in s).
Keeping proximity i.e. the number of times (per hour) the hen joins the
ducklings or the ducklings join the hen.
Disrupting proximity i.e. the number of times (per hour) the hen moves
away (> 1 m) from the ducklings or the ducklings move away from the
- Leadership: the number of times (per hour) the hen follows ducklings or
the number of times (per hour) the ducklings follow their mother.
Vigilance of the hen:
- Alert behaviours: the hen raises her head or inclines the head with a
- "Motionless Awake-Upright" behaviours: the hen remains upright without
moving and observes its surrounds.
Avoidance: one animal moves away when another one approaches.
Pecking: one animal pecks another one.
These behaviours were recorded whenever they occurred between the
hen and offspring or between the ducklings. In addition, to evaluate the strength
of cohesion between the ducklings, gathering or dispersion were also noted. The
ducklings were considered to be gathered when the distance between them did
not exceed 50 cm, and otherwise as dispersed.
In fact, each of these categories of behaviours will enable the
determination of the intensity of the parental care each week. If the hen is mainly
involved in brood care, she will have less time to care about herself. This
probably means that self-maintenance behaviours will vary through the rearing
period, being less frequent in the first weeks after hatching and more frequent
close to fledging. The intensity of the hen-brood bond will indicate how much
the hen takes care of the ducklings by keeping close proximity with them. When
parental care decreases, then the hen will probably stay more distant from
offspring. Vigilance behaviours are involved in the protection of ducklings all
the time since they will be vulnerable to predators. Lastly, agonistic behaviours
are the sign of an increase in the independence of the ducklings, which implies
that the hen could decrease her attention towards them.
The duration of behaviours such as foraging, preening, alert, "Motionless
Awake-Upright", the distant or contact duration between the female and the ducklings
and the gathering/dispersion duration of the ducklings were timed in s and summed on a
weekly basis. The duration of observations each week and for each family was equal (5
x 40 min). Specific behaviours such as leadership, maintenance of the proximity when
the female or the ducklings move nearer or further away, pecking and avoidance were
expressed in frequencies (n/hour) and averaged on a weekly basis. Average body mass
and hormone concentrations were calculated on a weekly basis.
Weekly differences were tested with a one-way ANOVA for repeated
measurements followed by a post-ANOVA Holm-Sidak test for normally distributed
data and after checking for homogeneity of variance (Sigmastat 3.0, SPSS Software).
When normality and homogeneity of variance failed, an ANOVA on ranks for repeated
measures (Friedman non-parametric test) was performed.
A principal component analysis (PCA) based on the correlation matrix (Minitab
13 software) was carried out on all behavioural parameters displayed by each female
over the 9 weeks post-hatching period. Scores resulting from the first axis were used as
an index of the intensity of parental care. Parental care was considered as completed
when the parental care index remained negative. ANCOVA using weeks as a covariable
was used to test for differences of parental care behaviour index and hormone
concentrations between females.
Least squares correlations were performed by using Sigma Plot SPSS (7.0).
Values provided are means ± SE. All statistical tests are two-tailed, and
probability levels < 0.05 were considered as significant.
Timing of breeding and moulting and changes in body mass.
Hatching occurred between April 15th and June 23th (Fig. 1) with brood sizes
ranging from 2 to 14 ducklings. All chicks fledged during week 9 post-hatching.
For the earliest breeding female, moulting of body feathers began on 21th May
and between 29th June and 7th July for the others. In all hens, this moult started before
offspring fledged but in the three latest breeding females (hatching in June), it was
initiated before the end of the parental care phase (Fig. 1). Moulting of wing feathers
started between mid-July and early September and well after parental care behaviour
Body mass of females did not change over post-hatching time (F8,46 = 1.40,
p = 0.19). Mean body mass of ducklings increased with age according to a sigmoid
curve, body mass gain being close to zero at fledging. At week 6 post-hatching, i.e.
when parental care was resumed (see below), body mass of ducklings reached
84.2 ± 9.6 % of the hen's body mass. At fledging, ducklings weighed 95.2 ± 0.6% of the
hen's body mass.
Nine of the 22 behavioural parameters recorded changed significantly (p < 0.05)
from hatching to fledging (Table 1). The time spent by the hen resting, preening and
being more than 1 m from ducklings as well as the frequency of ducklings pecked by the
female increased by 3, 3, 6 and 20 fold respectively during the post-hatching period
(Figs. 2a-d). Conversely, the leadership behaviour of the female and of ducklings as
well as the time spent by the hen in close proximity with the ducklings decreased with
the post-hatching time reaching low values by week 6 (Figs. 3a-c). The frequency of
duckling approaches towards the hen and the duration of gathering among the ducklings
decreased after week 1 and 6, respectively (Figs. 3d,e).
Alert behaviour of the female tended to change significantly within the 9 weeks
post-hatching (F8,46 = 1.90, p = 0.08). It remained at a high level until week 4
post-hatching (range = 466 ± 69 to 568 ± 93 s/h), then decreased to values ranging from
444 ± 129 to 234 ± 57 s/h).
From the first week, at the moment of its release, the hen always vocalized. This
behaviour ceased after week 6 post-hatching.
The first axis of the PCA, representing the parental care index, accounted for
50% of the variation of all the behavioural variables displayed by the females. It was
negatively correlated with sleeping, preening, distance (>1 m) and female pecking
(variable loads between -0.42 and -0.25), and positively correlated with contact, female
leadership and following (variable loads between 0.34 and 0.40). This parental care
index changed over the post-hatching period (F8,46 = 16.34, p < 0.001). It decreased
linearly with time post-hatching (Fig. 4a), the mean reaching negative values from week
5 (individual range: week 4 to 6). There was no difference in parental index pattern
between hens (F6,47 = 0.09, NS). Parental care duration and strength were not related to
the brood size nor to the hatching date (F1,6 < 1.48, NS). Mean parental care index
decreased together with duckling body mass (r² = 0.98, F1,8 = 271.04, p < 0.001)
Plasma testosterone concentration always remained under the threshold of
detection (0.1 ng/ml). Plasma corticosterone concentrations ranged between 33.2 ± 7.7
and 53.7 ± 8.0 ng/ml and did not change over time post-hatching (F12,62 = 0.88, NS).
According to post-hatching time, prolactinemia did not differ between females
(F6,47 = 0.97, NS) but changed significantly (F12,70 = 4.92, p < 0.001) over time during
the first 13 weeks. Prolactinemia was maximal during the first week (49.7 ± 2.4 ng/ml),
and significantly different from values at weeks 7 to 13 (t12 > 3.51, p < 0.001). High
levels (34.2 ± 5.8 to 40.3 ± 3.9 ng/ml) were maintained until week 6 (Fig. 4b). Plasma
prolactin concentrations were then depressed by 36% on average and remained
unchanged until week 13, ranging from 21.5 ± 4.8 to 30.9 ± 5.3 ng/ml.
Prolactin concentrations of the first 6 weeks post-hatching, i.e. corresponding to
the parental care phase, were not related to date (julian days, F1,40 = 0.81, NS), but
values from week 7 significantly decreased with date (r² = 0.46, F1,73 = 63.26,
p < 0.0001). In all females, the lowest plasma levels of the reproductive season
(11.4 ± 0.7 ng/ml) were reached between the 1st and 18th of August, independently of
the hatching date, with inter- and intra-individual variability in the plasma prolactin
concentration being reduced five-fold.
Hormones and behaviour
Mean weekly values of the parental care index were related to the mean weekly
prolactin concentrations according to a sigmoide curve (r² = 0.81, F3,8 = 7.03, p < 0.05,
Fig. 5). The shape of the relationship between the two parameters was similar to that of
a dose-response curve, the index of parental care increasing abruptly for plasma
concentrations of prolactin ranging between 34 and 40 ng/ml. Logarithmic transformed
mean prolactinemia and duckling body mass were negatively correlated (r² = 0.60, F1,8 =
13.07, p < 0.01). There was no relationship between prolactin and corticosterone
concentrations (F1,12 = 1.14, NS).
From the study two major results emerged: 1) there is a progressive decline of
parental care during the 6 weeks post-hatching, followed by a non-parental care phase,
until fledging, 2) there is a positive relationship between this parental care behaviour
and prolactinemia that remains at a high level for several weeks after hatching.
Overall, our data obtained in laboratory conditions give new and fundamental
evidence to confirm and understand those originating from field observations in ducks
and in other precocial species i.e. the degree of female attentiveness toward her brood is
the highest within the first two weeks after hatching (Kear, 1970; Afton and Paulus,
1992; Sherry, 1981; Richard-Yris et al., 1988) before brood abandonment after 6-7
weeks post-hatching (Ringelman et al., 1982; Talent et al., 1983). However, for
hatching occurring between mid-April and late June, we show that the strength and
duration of parental care behaviour 1) was not enhanced by the fact that hens and their
brood were maintained in close, forced proximity for up to 13 weeks, 2) was not lower
in late than in early breeding females and 3) did not support the brood-size hypothesis
which suggests that females attending larger broods provide more care to their
offsprings (see Pöysä et al., 1997 for a review). This latter result is in accordance with
that on brood abandonment fates reported for other dabbling ducks (Gendron and Clark,
2000) and with that reported by Pietz and Buhl (1999) who found no relation between
the hen behaviour and brood size in wild free ranging mallards. Overall, we agree with
the model developed by Lazarus and Inglis (1986) expecting that, especially in
precocial birds, parental investment remains independent from brood size if the
investment benefits all ducklings simultaneously and if predators do not take the entire
brood when they attack.
The strength of parental care behaviour has been evaluated in some precocial
species from the alert behaviour of the female, based on the fact that in species like
pink-footed geese, Anser brachyrhynchus, and in wild free-ranging mallards, parents
rearing a brood spent much more time being alert than those without young (Lazarus
and Inglis, 1978; Pietz and Buhl, 1999). From our data this behaviour was observed with
the highest frequency during the four weeks post-hatching and tended to decrease
afterwards, but non-significantly. In agreement with this latter observation, changes in
time spent alert according to the brood age gave controversial results among duck
species (see Pietz and Buhl, 1999 for a review). This suggests that the analysis of the
alert behaviour alone (or more generally of a single behaviour) is not sufficient to
evaluate precisely the strength of parental care. Indeed, of the 22 behavioural
parameters we recorded, 40% (pertaining to 3 of the 4 behavioural categories) were
significantly modified during the rearing period. In agreement with the leadership
character of hen and ducklings, females spent most of their time being close to the brood
during the first 5 weeks post-hatching. This was at the expense of the time allocated to
self-maintenance behaviours such as preening and resting. From week 6 post-hatching,
females clearly expressed an agonistic behaviour toward the brood and the duration of
their self-maintenance behaviours increased. At that time the frequency of approach
attempts by the brood towards the female was also lowered and, interestingly, the
ducklings gathered less. Altogether, these behaviours that developed during the
non-parental care period suggest a kind of reciprocal intolerance between the different
individuals of the hen-brood unit. To sum up we show here that the hen-brood bond as
well as its disruption (here at week 6 post-hatching) appears to be a complex
phenomenon requiring a complete behavioural analysis to be fully characterized.
In birds such as ducks, females have to moult before the fall migration. This
could lead to possible energy and nutritional conflicts with brood rearing at least in late
breeders (Ringelman et al., 1982; Hohman et al., 1992). In our study, body moult (along
the sides and flanks) that begun globally in early July, whatever the age of the brood, did
not impair the parental care behaviour, and vice versa. Conversely, the intense moult of
flight feathers was always initiated after the disruption of the parental care period. This
latter result is consistent with the idea of conflicts in nutritional requirement between
rearing young and wing moulting (Hohman et al., 1992).
Prolactin and parental care.
In precocial birds including ducks, prolactin is involved in parental care and nest
attentiveness during incubation (Goldsmith, 1991; Hall, 1991; Criscuolo et al., 2002).
In female mallards, mean circulating prolactin concentrations have been shown to
increase from about 30 ng/ml to 50-55 ng/ml throughout the course of incubation before
dropping sharply immediately after hatching (Goldsmith and Williams, 1980; Bluhm
1992; Hall, 1987). Thus, conversely to altricial species, this hormone was generally
considered not to be involved in parental care during young-rearing in precocial birds
(see Goldsmith, 1991 for a review). However, results on the Spotted Sandpiper, Actidis
macularia, the Wilson's Phalarope, Phalaropus tricolour, the Bar-headed Goose, Anser
indicus, the Common Eider and the Bantam hen, Gallus domesticus, mitigate this
assumption (Dittami, 1981; Oring et al., 1986, 1988; Sharp et al., 1988; Criscuolo et
al., 2002). In those precocial species, although prolactin concentration is set at a lower
level than during incubation, it remains higher than in adults not rearing young. This Download full-text
fact suggests that this hormone is likely involved in parental care after hatching
(Criscuolo et al., 2002). By analysing concomitantly the behaviour and the hormonal
profile of the hen we show here that in a precocial species, a well-defined relationship
between parental behaviour and prolactin release was present throughout the duckling
rearing period. This decline of prolactinemia concomitant to the hen-brood bond
disruption, gives strong arguments for a role of prolactin in promoting and/or
maintaining post-hatching parental care as demonstrated for altricial species. Moreover,
the index of parental care was correlated with prolactin concentrations according to a
sigmoide curve (Fig. 5), suggesting that this behaviour may only be stimulated once
plasma prolactin concentrations exceed a definite threshold level. Additionally, the
contribution of other factors such as the activity and the growth of the ducklings may not
be excluded. External stimuli are important factors stimulating prolactin secretion
which in turn increase readiness to brood care. In their experiments of nest deprivation
Sharp et al. (1988) showed a decline in plasma prolactin in the hen when not given
chicks, suggesting that chicks may stimulate or at least maintain to some extent
prolactin levels for some period of time after hatching. In the same way, changes in
duckling growth and behaviour could intervene in the control of prolactin secretion and
consequently contribute to stimulate the ending of parental care. Firstly, the mean body
mass of the six week old ducklings was about 84% of that of the females and, at this age
their morphological aspect (size and plumage) is close to that of the hen (Gollop and
Marshall, 1954, Anonymous, 1982). Secondly, considering that ducklings become
independent from week 6 by remaining distant from the hen, together with the female
intolerance towards ducklings, suggests that the activity changes of ducklings may
enhance hen-brood bond disruption through the prolactin secretion pathway (see also