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Physiology & Behavior 91 (2007) 229 – 239

Ontogeny of vocalizations and movements in response


to cooling in chickens fetuses
Albin Gräns, Jordi Altimiras ⁎
Department of Biology, IFM, University of Linköping, SE-58183, Sweden
Received 13 October 2006; received in revised form 27 February 2007; accepted 5 March 2007

Abstract

Bird incubation demands a balance between parental needs for foraging with fetal needs for heat provision and protection so that any means of
communication between the fetus and the parents would have an adaptive value. The aim of the study was to investigate whether putative avenues
of feto-maternal communication would correlate to physiological changes caused by environmental alterations. Oxygen consumption was used as
an indicator of fetal well being. The frequency, duration, intensity and composition of fetal vocalizations and the frequency and intensity of
movements were used to evaluate the potential for communicating fetal status quo. Fetuses of broiler chickens (Gallus gallus domesticus) at three
developmental stages (day 18, internally pipped and externally pipped) were challenged by a stepwise reduction in ambient temperature down to
30 °C. A drop in oxygen consumption in response to lowered temperatures was found in all stages. No differences correlating with temperature
variations were found in any of the variables associated with fetal vocalization, even if externally pipped fetuses vocalized more than internally
pipped fetuses. Movement occurrence and movement intensity, however, increased initially and decreased at temperatures below 35.0–35.5 °C.
Considering that the lower limit of optimal development is between 35 and 36 °C, the results suggest that fetal movements can be of potential use
to the incubating parent to assess and protect the well-being of the fetus.
© 2007 Elsevier Inc. All rights reserved.

Keywords: Feto-maternal communication; Prenatal; Oxygen consumption; Fetal vocalization; Fetal movement; Development

1. Introduction incubation, the incubating parent must find a balance between


their own need to obtain energy by foraging, with the thermal
During the early evolution of birds, development of a and protective needs of the fetuses [4]. Consequently, parental
higher body temperature would have increased incubation knowledge on the status and well-being of the fetus has a
temperature which, in combination with egg–parent contact beneficial value. On one hand, feto-parental communication
incubation, led to a higher hatching success [1]. Higher egg could proceed via vocalizations, movements or even chemical
temperatures reduce the time of incubation [2] and conse- signaling, particularly during late in ovo development, but these
quently the risk of predation. Selection pressure probably mechanisms have not been extensively studied. On the other
strengthened and further improved contact incubation beha- hand, parental attendance can be also driven by temperature
viours that allowed a better control of the developmental sensitive mechanisms arising from the brood patch [5]. The
environment. present study focused on the former, and analyzed the
Newly laid avian eggs are ectothermic and cannot control correlation between possible avenues of feto-maternal commu-
their own temperature without external aid [3]. During natural nication with the physiological status and well being of the fetus
when exposed to cooling.
Domestic chickens of a broiler strain (Gallus gallus
⁎ Corresponding author. Biology/IFM, Linköpings Universitet, 58183 Lin- domesticus) were chosen as test subjects. Broiler strains are
köping, Sweden. Tel.: +46 13 285824; fax: +46 13 281399. selected for fast growth and known to, in adult life, have
E-mail address: jordi@ifm.liu.se (J. Altimiras). problems to cope with high temperatures [6].
0031-9384/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.physbeh.2007.03.002
230 A. Gräns, J. Altimiras / Physiology & Behavior 91 (2007) 229–239

Fig. 1. Procedural flowchart for the analysis of fetal vocalizations. The data displayed is just a small segment of the real segment analyzed.

Fetal well-being was assessed by measuring oxygen con- studies on temperature related vocalizations prior to hatching
sumption as indicator of metabolic rate while reducing have yielded contradictory results [11].
temperature in the incubator. Oxygen consumption (VO2) is Although the ontogeny of prenatal [12] and postnatal [10]
affected by cooling as has been shown in egg-laying chicken behaviours has been described qualitatively, we aimed for a
strains [7,19]. thorough characterization of fetal movements and vocalizations
Simultaneous to the measurement of metabolic rate, prenatal that would include their frequency and intensity but also the
vocalizations and movements were recorded as potential qualitative composition of the potential message.
avenues of communication from the fetus to the parent [8]. The focus on the behavioural part of the study was to test
Vocalizations in hatchlings are important for communication of, whether fetal vocalizations and fetal movements would be
among other aspects, the need of heating assistance [9,10], but affected in response to cooling. We predicted that all variables
A. Gräns, J. Altimiras / Physiology & Behavior 91 (2007) 229–239 231

associated with fetal vocalizations and fetal movements would using a multiplexer (TR-RM8 Respirometer Multiplexer, Sable
increase as the temperature dropped and that the responses Systems International, Las Vegas, USA). The chamber not used
would increase as time of hatching approached. for sampling was flushed using an air pump (Tetratec AP80, 80 l
h− 1, Tetra, Germany).
2. Materials and methods Push-mode respirometry was used because it is less
sensitive to leaks [15]. In push-mode respirometry the oxygen
2.1. Subjects of study concentration ([O2]) of the air is measured after being pushed
through the chamber containing the oxygen consuming
Experiments were conducted on fertile broiler chicken eggs sample and compared with air pushed through a baseline
(Gallus gallus domesticus) of the strain Ross 308 obtained from chamber [15]. To avoid dilution effects by the presence of
a local hatchery (Lantmännen SweHatch AB, Väderstad, water vapor, the air sample was dried through a dessicator
Sweden). Prior to incubation the eggs were stored at a constant column (10 ml of indicating drierite, anhydrous calcium
temperature of 15 °C and automatically turned once every 4 h sulfate mixed with cobalt chloride, W.A.Hammond Drierite
(Hova-bator automatic turner, Savannah, USA). Fetal develop- company Ltd., Xenia, USA). The Multiplexer and the FOX II
ment was monitored by candling the eggs weekly and removing Analyzer were connected and controlled by a PC-computer
non-fertile and non-developing eggs. (Dell Optiplex GXa). Oxygen levels, atmospheric pressure
On day 0 the eggs were weighed to the nearest hundredth of a and flow were sampled at 0.1 Hz and analyzed using a custom
gram (BP 221S, Sartorius AG, Germany) and placed in a forced made data acquisition program (Lab View 8.0, National
draft incubator (25 HS, Masalles Commercial, Spain) set at Instruments, USA).
37.8 °C and 45% RH. The eggs were automatically turned once To prevent artifactual readings between the experimental
every hour until day 19 when they were placed in a hatching chambers due to incomplete wash-out of the respirometry
tray in the bottom of the incubator. The average incubation chamber, a wash-out test was conducted. A respirometry
length for domestic chicks of Ross 308 is 21 days. chamber was flushed with nitrogen until oxygen concentration
Fetuses at three different developmental stages were used in [O2] read zero and then it was flushed with air from inside the
the experiments: 18 days after incubation started (18d), experimental incubator at 50 ml min− 1. The test was stopped
internally pipped (IP) and externally pipped (EP). IP and EP when [O2] reached stable values. A 95% flush was achieved
are better markers than fetal age because they pinpoint a crucial after 6 min 20 s. Based on these results the respirometry
event for fetal maturation such as the onset of lung breathing chambers were sampled for 15 min at the end of each 30 min
[7]. At the same time, the time of hatching can vary temperature interval but only the last 5 min were used. VO2 was
considerably [13] so the physiological status of the fetus is calculated as flow (ml min− 1) multiplied by the difference in
better judged by the pipping state. To determine whether or not oxygen concentration (ml O2 ml− 1 air) between the baseline
an egg was internally pipped, a 1-cm2 piece of the shell above chamber and the experimental chamber.
the air cell was removed under sterile conditions and replaced The VO2 data set was split into two subsets: subset 1 — data
by a microscopy cover glass that was subsequently glued to the from T1, T2 and T3 and subset 2 — data from T3, T4 and T5. A
shell (Super epoxy 2 component glue, Loctite, Sweden). The linear slope was calculated for each of the two subsets as an
operation was conducted on day 16 to avoid disturbance to the estimate of fetal metabolic temperature sensitivity and was used
air cell gas composition which could influence the time of to compare temperature sensitivity between the developmental
hatching [14]. During the operation caution was made so that stages and temperatures.
only the microscopy cover glass and the shell came in contact
with the glue and all glued eggs continued to develop normally. 2.3. Sound recording and analysis
IP eggs were used within 1 h after the onset of internal pipping.
All procedures involving manipulation of animals were A condenser microphone (∅ = 4.5 mm, Veco Vansonic,
approved by the local ethical committee of animal research PVM-6027) was inserted through a hole drilled into the wall of
(Dnr.27-06). the chambers and connected to the sound card of a PC
-computer (Dell Optiplex 17OL). Sounds were recorded for
2.2. Oxygen consumption and analysis 15 min at a sampling rate of 11025 Hz with an 8 bit resolution at
the end of each of the temperature intervals. A custom-made
Oxygen consumption was measured by open respirometry in data acquisition software program (Lab View 8.0, National
a push-mode configuration. Two chambers were used for the Instruments, USA) was used for digital storage. From the
tests: one containing a non-fertilized egg, which was used for recordings, the variables associated with fetal vocalizations
baselining, and one containing the experimental egg. The were extracted. Recordings were taken from IP and EP fetuses
chambers were equipped with two sets of tubing (Tygon R3603 but not on 18 fetuses since vocalizations are only possible after
3.2 × 4.8 mm) leading air in and out with a controlled flow of internal pipping [11].
50 ml min− 1 (FOX II Analyzer, Sable Systems International, Analysis of the sound recordings was conducted using
Las Vegas, USA). In the lid of the chambers, small fans (Papst, Adobe Audition 1.0.
25 × 25 mm, 3.2 m3 h− 1) were installed to increase air mixing. For a schematic description of the sound analysis pro-
Air flow was switched between the two chambers every 15 min cedure see Fig. 1. The sound data file was a mixture of
232 A. Gräns, J. Altimiras / Physiology & Behavior 91 (2007) 229–239

Fig. 2. Procedural flowchart for the analysis of fetal movement. The data displayed are just a small segment of the real segment analyzed.

background noise from the incubator and vocalizations (calls) the microphone and the sound source was not fixed as the fetus
from the fetuses. To improve the signal-to-noise ratio a noise (egg) moved during recordings.
profile was constructed from a call-free section of the sound
record. The noise profile was used to remove most of the Rreadings ððx−minÞ=ðmax−minÞÞ
Sound Intensity ¼ ð1Þ
background noise (step 1, Fig. 1). A band-pass filter fitted to number of readings
the hearing bandwidth of G. gallus [16] removed all other
unwanted frequencies (step 2, Fig. 1). The amplitude (dB) of
the faintest call produced by each fetus after noise removal x (point value) ^2⁎1000 (using all points greater than the
and filtering was used as the minimum threshold for auto- threshold)
matic detection provided the signal exceeded the threshold for min lower threshold ^2 ⁎1000
more than 1 ms (step 3, Fig. 1). In the last step, all silence max highest recorded value for this specific egg ^2⁎1000
periods were removed to simplify further analysis (step 4,
Fig. 1). In a second phase of the analysis, the sound events detected
The average sound intensity for all readings between the were analyzed to distinguish between consecutive sound events
minimum and maximum values was normalized relative to each that formed a note and consecutive notes that formed a call [9].
fetus' specific settings and calculated by using Eq. (1). The To set up a consistent criterion to distinguish sound events into
more common RMS (Root Mean Square) method to measure notes and calls, the frequency distribution of the interval
signal-strength could not be used because the distance between between automatically detected sound events was analyzed.
A. Gräns, J. Altimiras / Physiology & Behavior 91 (2007) 229–239 233

Two thresholds were chosen, a short one defining a note and a


longer one defining a call. All silence periods longer than the
call threshold indicate the start of a new call.
Mean phrase duration was calculated by dividing the time
left after removing all silence periods by the number of calls in
that interval (step 4, Fig. 1).

2.4. Movement recording and analysis

Within the respirometry chamber, the egg was placed on a force


sensor (Micro switch, FSL05N2C, force range 1500 g, average
sensitivity 0.024 mV g− 1, Honeywell, Canada). The force sensor
was connected to a low level D.C.Amplifier and a D.C.Driver
amplifier (Models 7P1 and 7DA respectively, Grass Instruments).
The amplifier was connected to a commercial data acquisition
system (ML865 PowerLab 4/25T Data acquisition System, AD
Instruments) running the software program Chart5 for Windows in Fig. 4. Schematic description of the trend analysis procedure. +/− = MAX trend,
−/+ = MIN trend, +/+ = POS trend and −/− = NEG trend (see Materials and
a PC-computer (Dell Optiplex 17OL). From the force recordings,
methods for further details).
the variables associated with fetal movements were extracted. Force
recordings were measured on all three developmental stages.
The recorded traces contained three sources of movement: out the movements originating from the fetuses, the data signal
large shifts associated with fetal movements detectable by the was filtered (2 Hz high-pass filter) and intensified as shown in
incubating parent, small periodic fluctuations related to Fig. 2 (step 1). The filter also removed all slow movements from
ventilation (not in 18d fetuses) and noise (unwanted move- the egg. All remaining values were then squared and amplified
ments) from the experimental incubator. To improve the signal- (step 2, Fig. 2). A minimum movement threshold was set for
to-noise ratio, remove ventilation and baseline drift and single each fetus and all episodes over the threshold were marked by
the analysis program (step 3, Fig. 2). Time between events and
event peak values were then used to calculate the occurrence of
movements and their intensity.
Fetal movement was recorded for 15 min at a sampling rate
of 1 kHz during each of the temperature intervals. From each
movement trace, two different variables were analyzed:
frequency and intensity.
The maximal intensity of movements of each egg was
normalized relative to the fetuses' specific settings and
calculated using Eq. (2).

Rreadings ððx−minÞ=ðmax−minÞÞ
Movement intensity ¼ ð2Þ
number of readings

x local maximum value of marked event.


min lower threshold = (Σ max − Σ min)/5))⁎1.5 (during 5 s
intensity ranges in low activity episodes of the first
15 s of each temperature interval)
max highest recorded value for a specific individual, with
the only exception that values more than 50 times
higher than the threshold were counted as events but
not as highest values

Table 1
Egg mass prior to incubation and fetal mass (without yolk sac) after trials
Stage N Egg mass (g) a Fetal mass trial day (g)
18d 10 67.0 (62.4–72.4) 33.7 (30.1–36.7) b
IP 10 66.7 (59.5–73.1) 39.9 (36.0–43.8)
Fig. 3. Experimental respirometry chamber (height 95 mm, ∅50 mm) consisting
EP 20 65.0 (56.3–76.6) 39.7 (33.8–43.1)
of the following elements: light plastic egg cup (a), force transducer (b),
∅4.5 mm microphone (c), female luer fittings with air inlet (d) and outlet (e), K- a
Values shown as mean (min–max).
type thermocouple (f), ∅25 mm fan (g) and O-ring (h). b
Significant lower fetal mass in 18 d compared with IP and EP.
234 A. Gräns, J. Altimiras / Physiology & Behavior 91 (2007) 229–239

2.5. Experimental protocol and temperature regulation

All variables were recorded simultaneously under five


different temperature settings labeled T1–T5 for convenience.
K-type thermocouples were fitted to the lid of the respirometry
chambers to record temperatures.
Step-wise temperature drops were obtained by decreasing
the temperature settings in the experimental incubator every half
hour. Each trial took 150 min and consisted of five 30 min
periods ranging from 40 to 30 °C. Each cooling step decreased
temperature in the chamber by an average of 2.5 °C. The
experiment started 1 h after the eggs were moved to the
experimental incubator to give them time to settle after handling
and manipulation. Each egg was used once and, at the
completion of the trial, the fetus was euthanized with an
overdose of pentobarbital (1 g kg− 1, Pentobarbital 100 mg/ml,
Apoteket, Sweden).
In the experimental incubator, the eggs were placed in
airtight Plexiglas respirometry chambers (inner dimensions:
height 95 mm, ∅50 mm) built to fit the size of broiler eggs (see
Fig. 3 for a pictorial description). To account for possible
discrepancies between chamber temperature and egg tempera-
ture due to the thermal inertia of the egg, a separate temperature
test was conducted where T-type thermocouples were inserted
1.5 cm into the eggs on day 15 in sterile conditions. Inspection
of the eggs, on the day of hatching, revealed that the tip of the
thermocouples was located in the allantoic fluid. The eggs were
treated in an identical manner to the eggs used in the
experiments. The procedure was noninvasive and all six fetuses
used in the temperature difference test developed normally to
the external pipping stage.

2.6. Statistical analysis

All statistical analyses were conducted in SPSS 12.0.1 for


Windows (SPSS Inc.)
VO2 differences between the three developmental stages
were tested using a general linear ANOVA model with stage as
main factor and temperature as a covariate. Paired comparisons
were done using Bonferroni's post hoc test.
To test for significant differences between fetal develop-
mental stages in all other variables measured, namely call
occurrence, call composition, call duration, average note
intensity, movement occurrence and maximal movement
intensity, a repeated measures ANOVA model was used with
developmental stage as between subject's factor and the
different temperature intervals as the repeated factor.

Fig. 5. (A) Oxygen consumption at different ambient temperature intervals (T1–


T5) in chicken fetuses of three different developmental stages: 18 days old fetuses
(shaded triangle = 18d, N = 10), internally pipped fetuses (open triangle = IP,
N = 10) and externally pipped fetuses (closed triangle = EP, N = 20). (B) Relative
oxygen consumption in response to cooling. (C) Temperature sensitivity in the
two different trend subsets and a temperature corrected version of subset 1.
⁎ indicates significantly higher temperature sensitivity compared with 18d
(p b 0.05). # indicates significantly higher temperature sensitivity between
CorrT1–T3 and T3–T5 (p b 0.05). Data as mean (SD).
A. Gräns, J. Altimiras / Physiology & Behavior 91 (2007) 229–239 235

Table 2 3.1. Oxygen consumption


Frequency distribution of the four possible trends
Variable Stage MAX MIN POS NEG χ2 a χ2-Yates b The VO2 response to gradual cooling on fetuses of the three
Oxygen consumption 18d 0 0 0 10 c, d
22.5 19.6 developmental stages studied is shown in Fig. 5A. For all
IP 1 0 0 9 c, d 16.9 14.4 developmental stages, the difference in VO2 between the highest
EP 0 1 0 19 c, d 39.2 36.5 and lowest temperature was above 60% (18d — 62.2%, IP —
Call frequency 18d – – – – – –
61.6%, EP — 65.5%, see Fig. 5B). All three developmental stages
IP 2 1 3 2 0.5 0.125
EP 4 6 3 4 0.72 0.37 showed a similar decline in VO2 as temperature decreased. The
Call composition 18d – – – – – – difference in VO2 between 18d and EP was 30.4% compared to
IP 4 1 2 1 2 1.13 the 15.1% difference in fetal mass. A significant amount of fetuses
EP 5 7 2 3 1.39 0.89 ( p b 0.05) showed the NEG trend for VO2 at all developmental
Call duration 18d – – – – – –
stages (Table 2).
IP 5 1 2 1 3.36 2.25
EP 3 6 5 3 1.00 0.56 The lowest metabolic temperature sensitivity occurred at 18d
Average sound intensity 18d – – – – – – 10.15 μl O2 min− 1 °C− 1 (2.13) in the first subset (T1–T3) and
IP 4 1 2 2 1.36 0.69 the highest at EP 32.63 μl O2 min− 1 °C− 1 (7.64) in the second
EP 3 8 2 4 3.31 2.49 subset (T3–T5). However, chamber temperature, for the
Movement occurrence 18d 6c 2 1 1 4.9 3.6
protocol adopted, is not a good indicator of embryonic
IP 6c 3 1 0 4.9 3.6
EP 4 0 2 4 0.9 0.4 temperature in the two first temperature steps (T1 and T2)
Max movement intensity 18d 7 c, d 1 1 1 8.1 6.4 because at temperatures over 35 °C, the chamber temperature
IP 4 2 0 4 0.9 0.4 decreased faster than the temperature inside the egg (Fig. 6). No
EP 4 2 1 3 0.9 0.4 differences were observed below 35 °C (Fig. 6). In conse-
a
χ2 value of the observed-expected comparison between the highest quence, a better estimate of temperature sensitivity in the T1–
occurring trend and the other trends (critical values 2.71 (p = 0.1) and 3.84 T3 subset was recalculated taking into account the temperature
(p = 0.05) when df = 1).
in the allantoic fluid by interpolation. As shown in Fig. 5C the
b
Observed-expected comparison χ2 value with application of Yates
correction. recalculated T1–T3 sensitivities are consistently more similar to
c
Significantly higher occurring trend. T3–T5 sensitivities. Only at 18d there was a significant
d
Significantly higher occurring trend after using Yates correction. difference between temperature sensitivity in the T1–T3 and
T3–T5 intervals ( p = 0.007).
Temperature sensitivity was significantly lower at 18d in
In the repeated measures ANOVA model, differences comparison with IP ( p = 0.010) and EP ( p = 0.000) in the T3–
between temperatures (i.e. temperature as a second between T5 range (see Fig. 5B) but not at temperatures above 36 °C.
subject's factor) could not be assessed because the temperature
treatment was sequential (progressive cooling) and not 3.2. Fetal vocalizations
randomized. Instead, an alternative trend analysis was used.
For trend analysis, data of all variables from all fetuses The distribution of time intervals between automatically
from all developmental stages was split into two subsets: detected sound events allowed an objective analysis of the
subset 1 — data from T1, T2 and T3 and subset 2 — data
from T3, T4 and T5. A linear slope was calculated for each of
the two subsets and the signs of those lines were combined to
define a trend. The four possible trends are shown in Fig. 4.
The MAX and MIN trends indicate the presence of a
temperature threshold. The POS and NEG trends occur
when the response is directly or inversely correlated to the
strength of the exposure respectively. A chi-square distribution
analysis was used to evaluate the frequency of the different
trends with the null hypothesis that an even distribution of
trends (25% each) should be expected if all trends were
equally likely to occur. To compensate for the existence of
only one degree of freedom, Yates correction was used [17].

3. Results

All data is presented as Mean (SD) [18].


As expected, fetal mass was significantly lower in 18d
fetuses in comparison with IP ( p = 0.000) and EP fetuses Fig. 6. Relationship between temperature of the respirometry chamber and egg
( p = 0.000) (Table 1). The difference in fetal mass between EP temperature of the allantoic fluid (N = 6). Data as mean (SD). The dotted line
and 18d fetuses was 15.1%. indicates the line of equality.
236 A. Gräns, J. Altimiras / Physiology & Behavior 91 (2007) 229–239

per call and call duration of 120 ms. The average sound
intensity was highest on IP fetuses during T3 [3.05 (3.41) a.u.]
and lowest on IP fetuses during T2 [0.50 (0.83) a.u.].
EP fetuses vocalized more than IP fetuses, as seen in a higher
frequency (F(1,28) = 5.657, p 0.024), longer duration (F(1,28) =
5.287, p = 0.034) and higher number of notes per call (F(1,28) =
15.61, p = 0.000). However, neither of the vocalization variables
showed a preference towards any of the four temperature trends
(Table 2).

3.3. Fetal movements

Fig. 9 shows frequency and maximal intensity of the


movement events of 18d, IP and EP at the different temperature
intervals. Frequency was highest for EP at T3 [3.53
(1.93) events min − 1 ] and lowest for 18d at T1 [1.71
(1.04) events min− 1]. The maximal intensity of movement
was found at T3 in 18d [10.05 (4.99) a.u.] and the lowest at T5
in IP [3.11 (2.06) a.u.] but no significant differences between
developmental stages were found for any of the fetal movement
variables.
A significantly higher number of fetuses showed the MAX trend
on movement intensity at 18d (pb 0.05, Table 2). Prior to using
Yates correction, which has been criticized for overcompensating
[17], the movement frequency at both 18d and IP also showed a
significantly higher frequency of MAX trends ( pb 0.05).

4. Discussion

4.1. Metabolic responses to decreasing temperatures

Fig. 7. Characterization of fetal calls. (A) Frequency distribution of the time VO2 values were in agreement with those from previous
duration between automatically detected sound events in IP-fetuses. The natural studies at normal incubation temperatures around 37.8 °C [7,15]
thresholds at 0.15 s and 0.8 s were used to define notes (N0.15 s) and calls and showed a strong temperature correlation typified by the
(N0.8 s) in the vocalization analysis. (B) Section of a sound signal with two NEG trend (see A, Fig. 6).
calls, x and y, containing 2 and 4 notes respectively. The silence between the two
The metabolic changes increased in magnitude with colder
calls (z), must exceed the time used to separate calls (N0.8 s). (C) Section of
sound record with one note. The time between values exceeding the threshold is temperatures and no metabolic compensation was found, unlike
shorter than the time used to separate notes (b0.15). earlier findings in egg-laying chicken strains [15,19]. Metabolic
compensation has been related to the appearance of incipient
vocalization recordings (histogram for IP fetuses shown in Fig. endothermic responses [7] but no significant differences in heat
7A, histogram for EP fetuses was very similar and is not production or Q10 were recorded at 18d, 19d and 20d chicken
presented). The trimodal distribution justified the definition of a fetuses exposed to temperatures ranging from 34 to 39 °C [20].
note as a series of consecutive sound events separated by less than VO2-based Q10 coefficients in the current study were 1.81, 1.84
0.15 s in between (Fig. 7C) and a call as a sequence of consecutive and 1.76 for 18d, IP and EP respectively.
notes separated by less than 0.8 s in between (Fig. 7B). After a While the presence or absence of metabolic compensation and
silent period longer than 0.15 s, a new note started. After a silent its thermoregulatory significance can be argued, no evidence of an
period longer than 0.8 s, a new call started and these calls could increase in metabolic rate during cooling has been reported in 17d
contain one or more notes. These definitions were consistent with to EP fetuses [7,15]. Instead, VO2 of 20d fetuses in ambient
a manual interpretation of the sound tracks: a note corresponded temperatures between 39 °C and 30 °C showed a VO2 drop of
to chirping sounds while a call was a cluster of notes. 33% [19], comparable to the 34.5% drop in the present study.
The effect of temperature on all vocalization variables: call The fetal metabolic response to cooling is in sharp contrast
frequency, call composition, call duration and average sound with the response in 1-day-old hatchlings, which increase VO2
intensity is shown in Fig. 8. The lowest frequency was found on by 36% when cooled from 39 °C to 33 °C [19]. Although it is
IP fetuses with the lowest interval average being 2 calls in appealing to speculate that heat production associated to the
15 min, 1.2 notes per call and a call length of merely 24 ms. The increased metabolic rate should mature during late in ovo
highest frequency was found on EP fetuses with the highest development the experimental evidence available in egg-laying
interval average of 9 calls during the 15 min period, 1.5 notes strains shows metabolic differences in some studies [15,19]
A. Gräns, J. Altimiras / Physiology & Behavior 91 (2007) 229–239 237

Fig. 8. Call-related variables: (A) frequency (calls per 15 min), (B) composition (notes call− 1), (C) average sound intensity (a.u.) and (D) and duration (s) at two
developmental ages at the five temperature intervals (T1–T5). Data as mean (SD). N = 10 (IP) and N = 20 (EP).

while not in others [7,20–23]. In absolute terms, temperature the absolute temperature sensitivity. When expressed as percent
sensitivity in this study correlated positively with fetal age and changes no differences in the slope of the temperature curves
negatively with temperature, i.e. the older the fetus the higher were apparent.

4.2. Behavioural responses to decreasing temperatures

Due to the lack of a self thermoregulatory response, fetuses


require parental attendance at the end of the incubation period to
maintain growth and facilitate hatching. It is in this situation that
the ability of the fetus to communicate its well-being to the
incubating parent, either via sounds or movements, would
acquire its maximal significance.
The vocalizations of hatchlings, juveniles and adult red
jungle fowl G. gallus have been extensively characterized into
two major groups: loud low frequency calls associated with fear
and distress and high frequency calls associated with pleasure
[9]. The vocalization repertoire in fetuses is more limited and
has been characterized following the same criteria that in adult
birds [8] even if the behavioural context of the call was not
readily available. Unlike earlier studies no attempts were made
to characterize the spectrogram of the recorded calls [8].
Instead, the study focused on quantitative variables related to
the strength and duration of the calls.
The significant increase in the frequency of calls, call
composition and duration between IP and EP is indicative of the
maturation in the vocalization ability of chicken fetuses as
hatching approached [8,11]. However, no temperature trends
were observed for any of vocalization variables at any of the
Fig. 9. Movement-related variables: (A) frequency (min− 1) and (B) maximal developmental stages, in agreement with earlier studies on
movement intensity at three developmental stages during temperature intervals domestic chickens G. gallus [11] and Muscovy Ducks Carina
T1–T5. Data as mean (SD), N = 10. moschata [24].
238 A. Gräns, J. Altimiras / Physiology & Behavior 91 (2007) 229–239

In many other bird species, however, egg cooling triggers A significantly higher number of fetuses responded with a
an increase in fetal vocalizations. For example, eggs of MAX trend on movement frequency at IP and both frequency
American Coot Fulica americana americana and Ring-billed and maximal intensity of the movements at 18d. The peak
Gulls Larus delawarensis vocalized more frequently when occurred at T3, corresponding to 35.5–36.0 °C (Fig. 9).
chilled [25,26] while Budgerigar eggs Melopsittacus undu- The most likely explanation of increased movement is as an
lates vocalized with longer calls at both higher and lower indicator of distress and would signal that temperatures around T3
incubation temperatures than control but with an increase in are important for fetal development. T3 temperatures are
frequency when the temperature increased [27]. Similarly, suboptimal while T2 temperatures correspond to the optimal
eggs of Eared Grebe Podiceps nigricollis vocalized more with range [4,13].
a decrease in the number of notes per call [28] and American Several studies dealing with the effects of ambient tempera-
White Pelican Pelecanus erythrorhynchos eggs made longer tures on avian incubation have proposed the existence of three
calls with both increased intensity and frequency when cooled important thermal thresholds: physiological zero temperature,
[29]. lower limit of optimal development and upper lethal temperature
Inter-specific differences in the ability to use vocalizations as [1,4,6,13]. The common temperature of naturally incubated
parental cues have been linked to the maturity at the time of avian fetuses is between 30 °C and 40 °C [13]. Consequently,
hatching. It has been argued that vocalizations are important in most birds will be exposed to the lower limit of optimal
precocial species, which are more developed at the time of development during natural incubation and a response evolving
hatching [30]. This is not a convincing argument since species just below the threshold of optimal development is possible. In
of different hatchling maturity types are represented in the chickens the lower limit of optimal development is 36.0 °C [1].
studies mentioned. While chickens, ducks, coots and grebes are In the current study, the significantly higher occurrence of
precocial, gulls are semi-precocial and budgerigars and pelicans the MAX peak trend at 35.2 °C (18d) and 35.5 °C (IP) is just
are altricial [31] so no particular pattern arises from that below the suggested temperature for the lower limit of optimal
classification. development, supporting the view that increased fetal move-
A more obvious pattern is found when considering the ment frequency and intensity can be parental signaling cues.
domestication history of the species. Studies conducted on eggs The MAX peak trend pattern was not apparent for EP
either in, or collected late from their natural incubation fetuses, implying that communicating heating assistance
environment show vocal responses to cooling: Budgerigars M. becomes less important closer to hatching. Supporting evidence
undulates [27], Eared Grebes P. nigricollis [28] Ring-Billed is found by the fact that cold challenged internally pipped
Gulls L. delawarensis [26] and American White Pelican P. chicken eggs (20 °C for 30 min) complete hatching at the same
erythrorhynchos [29]. On the other hand, studies on commercial time than control eggs [11].
species such as ducks [24] and chickens [11] showed no In summary, the main results of the study indicate that egg
increase in the vocalization responses to different temperatures. cooling imposes a decrease in metabolic rate in late development
However, the suggestion that domesticated species do not without indication of compensatory thermoregulation. At the same
respond to cooling with increased vocalizations cannot be time, EP fetuses vocalized more than IP fetuses but none of the
substantiated without further experiments because those studies vocalization variables correlated with temperature. Frequency of
were also carried out in conditions of artificial incubation. Vocal fetal movements in 18d and IP, but not EP, peaked at temperatures
interactions between mother and fetuses are present in between 35 and 35.5 °C, which are below the temperature for
domesticated White Leghorn chickens [8] and could be relevant optimal development in chickens. Fetal movements could serve as
for the development of a full vocalization repertoire, irrespec- potential parental cues to signal thermal distress because they
tive of the need to respond to ambient fluctuations. correlate with the degree of cooling. Fetal vocalizations might also
The continuous contact between the incubated eggs and the be used to signal thermal distress but the lack of correlation with the
parents' brood patch [13] is likely to allow the incubating parent degree of cooling and the quick maturation with fetal age makes
to detect movements within the eggs and thereby react to them them less informative for the attending hen.
by e.g. rearranging the eggs for a more even heat distribution.
Even so, several occasions of birds incubating dead eggs have Acknowledgements
been observed [4,32].
The absolute frequency of fetal movements is expected to The authors acknowledge Lantmännen SweHatch AB for
increase as hatching approaches because internal and external egg supply. Financial support was obtained from ELFA
pipping requires fetal movements. However, no significant Forskningsstiftelse and FORMAS (Dnr.22.1/2003-0987 and
differences in movement frequency between stages were found, Dnr.221 2004 1331).
most likely due to the constraints of the force recording system.
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