Thermogenic and Vocalization Responses to Cold in the Chicken Hatchling During Normoxia and Hypoxia

Thermogenic and Vocalization Responses to Cold in the Chicken HatchlingDuring Normoxia and Hypoxia

Khalid Al AwamMcGill University, King Saud University, and Prince Sultan

Cardiac Centre, Riyadh, Saudi Arabia

Florin Catana and Jacopo P. MortolaMcGill University

We investigated the vocalization and the thermogenic responses to cold during hypoxia in chickenhatchlings during the first postnatal day. Calls were quantified in number and sound characteristics(amplitude and frequency); the change in oxygen (O2) consumption, measured by an open-flowmethodology, represented thermogenesis. The cold challenge consisted of a decrease in ambient tem-perature (Ta) from �39 to 28 °C, in steps of 2 °C, or an acute exposure to �28 °C, either in normoxiaor hypoxia (10% O2). Hypoxia lowered thermogenesis and the critical Ta, suggesting a decrease in theset point for thermoregulation. The vocalization response to cold was rapid; did not progress with theduration or intensity of the cold stimulus; was similar in very young (�8 hr old) and older (12–24 hr)hatchlings despite their differences in thermogenic capacity; and was essentially unaffected by hypoxia.We conclude that the hatchling’s vocalization in the cold follows a stereotyped pattern not related to thethermogenic regulation of body temperature. The dissociation between vocalization and thermogenesismight carry some advantage in conditions of cold and hypoxia.

Keywords: birds, body temperature, oxygen consumption, thermogenesis

When confronted by a cold stimulus, mammals and birds com-bine behavioral and autonomic responses aimed to reduce heatdispersion (thermolysis) and increase heat production (thermogen-esis). In the cold, behavioral means usually operate to decreasethermolysis, for example, by finding a proper shelter, by assuminga posture that reduces the body surface exposed to cold, or bygrouping in a huddle. Autonomic responses are involuntary en-dogenous mechanisms under sympathetic control. They operateboth to reduce thermolysis through peripheral vasoconstriction andto increase thermogenesis, as with shivering and brown fat ther-mogenesis. A behavioral reduction in heat loss is usually lesscostly than any form of endothermic heat production; hence, in thecold, in addition to autonomic responses, birds and mammals makeextensive use of behavioral means for the control of body temper-ature (Bicego, Barros, & Branco, 2007). One may expect somedegree of reciprocity between the autonomic and behavioral re-sponses to cold (Weiss & Laties, 1961; Schlader, Prange, Mick-leborough, & Stager, 2009). However, behavioral thermocontrol isoften difficult to quantify, and little is known about its response tohypoxia.

Hypoxia inhibits the thermogenic response to cold (Mortola &Gautier, 1995). This is a regulated response and not simply theeffect of a limitation in oxygen availability. In fact, during hypoxichypometabolism, physiological or pharmacological mechanisms

are capable of raising oxygen consumption at or above the nor-moxic level (Mortola, 2004). In addition, during hypoxia, rats andother animals opt for an ambient temperature (Ta) that minimizesenergy expenditure (Wood, 1991; Gordon & Fogelson, 1991;Dupre & Owen, 1992) and increases heat loss (Tattersall & Mil-som, 2003; Scott, Cadena, Tattersall, & Milsom, 2008). Thisbehavior has been interpreted as an indication that hypoxia shiftsthe set point for the control of body temperature downward (Mor-tola & Gautier, 1995).

Newborn mammals and birds rely heavily on behavioral meansfor the control of their body temperature (Tb). In fact, at birth,newborns have minimal shivering capacity and only some degreeof nonshivering thermogenesis, which improves with time in thepostnatal period (Hey, 1969; Alexander, 1975; Blix & Steen, 1979;Szdzuy, Fong, & Mortola, 2008; Mortola, 2009). Like in the adult,in newborns, hypoxia depresses autonomic thermogenesis (Mor-tola & Gautier, 1995), but how this interacts with behavioralcontrol has rarely been considered. Young guinea pigs in a ther-mocline during hypoxia chose a lower Ta than in normoxia; theirresponse to cold was not investigated (Clark & Fewell, 1996).Newborn rats in the cold huddled less in hypoxia than duringnormoxia, in agreement with the concept of the drop in the Tb setpoint during hypoxia (Mortola & Feher, 1998). Whether or notvocalization can be considered a behavioral mechanism of defenseagainst changes in environmental conditions is questionable. Infact, from experiments performed in neonatal rats during cold, ithas been argued (Blumberg & Alberts, 1990) that the soundproduction is the unavoidable by-product of some degree of nar-rowing of the vocal folds (or laryngeal braking). This is a mech-anism that neonates of many species utilize, probably to improvethe intrapulmonary distribution of airway pressure, lung expan-sion, and gas exchange (Mortola, 2001). In such a case, eventhough they can elicit a maternal response, these neonatal calls

Khalid Al Awam, Department of Physiology, McGill University, De-partment of Physiology, King Saud University, and Prince Sultan CardiacCentre, Riyadh, Saudi Arabia; Florin Catana and Jacopo P. Mortola,Department of Physiology, McGill University.

Correspondence concerning this article should be addressed to Jacopo P.Mortola, McGill University, Department of Physiology, room 1121, 3655Sir William Osler promenade, Montreal, Quebec, H3G 1Y6 Canada.E-mail: jacopo.mortola@mcgill.ca

Behavioral Neuroscience © 2011 American Psychological Association2011, Vol. 125, No. 1, 74–83 0735-7044/11/$12.00 DOI: 10.1037/a0021953

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would have an autonomic origin. Whether this interpretation ap-plies to all forms of neonatal vocalization, including that of hatch-lings in cold and hypoxia, is not known. Nevertheless, irrespectiveof their primary origin and purpose, distress calls are an effectiveresponse to cold because they attract parental attention withoutmajor energetic cost.

In birds, calls are effective thermobehavioral mechanisms evenbefore hatching (Evans, Whitaker, & Wiebe, 1994; Brua, Nuech-terlein, & Buitron, 1996). In this study, we have examined howvocalization and autonomic thermogenesis respond to cold andhypoxia, singly or combined. We have used the chicken hatchlingas an animal model because the vocalization of the hatchling,being relatively stereotyped (Bugden & Evans, 1991, 1999; Marx,Leppelt, & Ellendorff, 2001), is more easily detected and quanti-fied than the sounds produced by neonatal mammals. The thermo-genic capacity of the chicken hatchling changes rapidly in the firsthours after birth (Szdzuy et al., 2008). Hence, it is possible toexamine to what extent vocalization in response to cold changes asautonomic thermogenesis increases with age.

Materials and Method

Animals

Freshly laid fertilized eggs of White Leghorn chickens (Gallusgallus) were obtained from a local supplier. The eggs wereweighed and placed in incubators (Hova-Bator 1602, Savannah,GA) around midday (embryonic day E0). The incubators main-tained a steady temperature of 38 °C, a relative humidity of 60%,and provided a 45o egg rotation 4 times a day. A data logger(HOBO, Onset Computer Corporation, Bourne, MA) inside theincubator monitored the incubation temperature and humidity ev-ery 10 min.

Experiments were conducted on hatchlings in the second half ofthe day of birth (E20-E21). A separate group of hatchlings wasstudied in the early postnatal hours (�8 hr; see Table 1). Theexperiments consisted of monitoring the thermogenic and vocal-ization responses to cold and hypoxia, singly or combined. Thethermogenic response was quantified from the increase in oxygenconsumption (VO2), and the vocalization response was quantifiedfrom the number and sound characteristics of the hatchling’s calls.One protocol consisted of progressive small-step decreases inambient temperature (Ta); the second consisted of the time courseof the responses to an acute exposure to cold. Both protocolsincluded runs in normoxia and hypoxia.

Tb was measured at the beginning and the end of the experimentby gently inserting a fine tungsten-constantan thermocouple con-

nected to a digital thermometer (Omega 871A, Omega Engineer-ing Inc., Stanford, CT) for a length of about 15 mm into the cloaca.

Oxygen Consumption

The hatchling was placed in a 300-ml plastic container main-tained at the desired temperature by a water bath. A steady gasflow (air or hypoxia) of 200 ml/min was continuously deliveredthrough the respirometer under the control of a precision needle-valve flowmeter. The inflowing and outflowing gases were sam-pled, passed through a drying column, and monitored by a cali-brated oxygen analyzer (OM-11, Beckman, Fullerton, CA). Theoutput of the analyzer was displayed on a computer monitor duringonline acquisition, and VO2 was computed from the flow rate andthe inflow-outflow gas concentration difference, averaged overeach 5-min time interval. Values of VO2 are expressed at standardtemperature pressure dry (STPD) conditions in ml/min.

The ambient temperature (Ta) of the respirometer was moni-tored by two thermocouples placed at the level of the hatchling. Tavalues were taken every minute for the whole duration of theexperiment and averaged over each 5-min time interval.

Sound Recording

A microphone incorporated in the respirometer, just above thehatchling, was used to monitor the hatchling’s calls. Mono-channelsound acquisition was obtained by use of commercially availablesoftware (WavePad Sound Editor®, NCH Software Inc., Green-wood Village, CO) at 44100 Hz.

On playback of the audio file (Audacity®, Free Software, Inc.,Boston, MA), the most convenient time-scale magnification wasused to discriminate the individual calls and to filter occasionalnoise. The number of calls (or pulse rate) was counted for each 5min of the experimental recording. Fast Fourier Transformationanalysis was conducted using the aforementioned program tocompute the sound amplitudes (dB) and frequencies (Hz) compo-nents of the calls; the latter represent the average of all the clearlydistinguishable peaks of the spectrum, including the dominantfrequency (the frequency containing the greatest amount of acous-tical energy). Data of amplitude and frequency were averaged overeach 5-min time interval.

Protocols

Two experimental protocols were followed on different groupsof hatchlings. Protocol #1 aimed to define the Ta-VO2 curve(thermogenic response), from which the lower critical Ta (Ta,below which VO2 increases) and the sensitivity to cold (slope of the

Table 1Hatchlings Used for Each Experiment

N Initial egg weight (g) Hatchling’s weight (g) Hatchling’s age (hr)

Protocol #1: Step ambient temperature changes 17 63.8 � 0.7 43.7 � 0.6 19.0 � 0.9Protocol #2: Acute cold, 12–24 hr old 17 59.9 � 0.9 41.9 � 0.6 16.9 � 1.2Protocol #2: Acute cold, �8 hr old 17 61.2 � 0.8 44.3 � 0.8 4.1 � 0.4

Note. Values are means � 1 SEM; g � grams.

75THERMOGENIC AND VOCALIZATION RESPONSES TO COLD

Ta-VO2 relationship) were computed and compared between nor-moxia and hypoxia. The same analysis was performed for the calls.Protocol #2 examined the autonomic and vocalization responses asfunction of time after an acute cold exposure.

Protocol #1: Step decreases in Ta. The hatchling was placedin the respirometer at 37.5 °C for 1 hr before slightly increasing thetemperature of the water bath to raise the respirometer Ta by about1 °C in 10 min. Then, the water bath was adjusted to lowerthe respirometer Ta by about 2 °C every 10 min, over the range39–29 °C. At each 10 min step, recordings were obtained duringthe last 5 min. Upon termination, the respirometer was broughtback to 37.5 °C, maintained at that Ta for 1 hr, and the wholeexperiment repeated in hypoxia. Hypoxia was obtained by con-necting the inlet of the respirometer to a gas-impermeable bagfilled with a 10% O2 mixture. The sequence of normoxic andhypoxic runs alternated among hatchlings. Tb was measured at thebeginning and the end of the experiment.

Protocol #2: Acute exposure to cold. After 1 hr at 37.5 °C,recordings were obtained for the following 15 min (warm condi-tion). Then, the respirometer was rapidly transferred to a secondbath preset at �24 °C. Recordings resumed 5 min later andcontinued uninterrupted for 30 min. Following the run in nor-moxia, the original warm conditions were reestablished and main-tained for at least half an hour, after which time the experimentwas repeated in hypoxia (10% O2). All hatchlings were subjectedto both runs, and the sequence of normoxia-hypoxia alternatedamong animals.

To examine the vocalization response of hatchlings with mini-mal thermogenic capacity (Szdzuy et al., 2008), a separate groupof young hatchlings, all aged between 1 and 8 hr, was studied witha protocol identical to the one just described. In all cases, Tb wasmeasured at the beginning and at the end of the experiment.

Data Analysis and Statistics

Data are presented as group means � 1 Standar Error of theMean (SEM), unless otherwise indicated. Comparison betweentwo sets of data was performed by two-tailed t tests. From theresults of Protocol #1, the VO2 sensitivity and the lower critical Ta(i.e., the Ta below which VO2 begins to rise) were calculated, firstby excluding the two lowest VO2 data points, then by applyinglinear regression to the remaining Ta-VO2 data points (see Resultssection). The slope of the regression represented the sensitivity ofVO2 for Ta. The lower critical Ta corresponded to the Ta value ofthe linear regression at which VO2 equaled the average of the twolowest VO2 values.

The statistical comparisons of the data in hypoxia and normoxiaat different values of Ta (Protocol #1) or at different times (Pro-tocol #2) were accomplished by two-way repeated measuresANOVA, one factor being Ta and the second factor being the O2

level, followed by post hoc Bonferroni’s limitations for all possiblecomparisons. Unpaired ANOVA was adopted for the comparisonof the responses of the two age groups of hatchlings. Specificsabout the statistical tests adopted on each occasion are provided inthe Results and in figure legends. Critical values of the correlationcoefficients, r, differences between slopes or between sets of datawere considered statistically significant at p � .05.

Results

Protocol #1: Stepwise Decreases in Ta

The Ta profile within the respirometer during stepwise cold wasapproximately linear, by �2 °C/10 min, and similar in normoxiaand hypoxia (see Figure 1).

The Ta-VO2 relationship was flat at the highest Ta, then VO2

increased with the drop in Ta (Figure 2, top). The lower critical Taaveraged 36.4 � 0.5 °C and the VO2 sensitivity, measured asdescribed in the Method section, was 0.12 � 0.01 ml O2/min per°C drop (dashed line in Figure 2).

In hypoxia, VO2 was less than in normoxia at all Ta values below37 °C ( p � .01; two-way repeated measures ANOVA). The lowercritical Ta (34.0 � 0.4 °C) and the VO2 sensitivity (0.066 � 0.01ml O2/min per °C drop) were both significantly less than innormoxia ( p � .0001 and p � .001, respectively).

The profile of the Ta-Calls curve was qualitatively differentfrom the Ta-VO2 curve, mainly in two aspects. First, the peaknumber of calls was reached at �36 °C and did not changesignificantly with further drops in Ta (Figure 2, bottom). Second,the Ta-Calls curve in normoxia did not differ significantly from thehypoxic curve at any Ta (two-way repeated measures ANOVA).

Tb in warm conditions averaged 39.6 � 0.2 °C. At the end of thecold runs, Tb was significantly decreased, more so in hypoxia(35.4 � 0.2 °C) than in normoxia (36.6 � 0.5 °C; one-wayrepeated measures ANOVA).

Protocol #2: Acute Cold Exposure

The time course of the temperature in the respirometer duringacute exposure to cold differed slightly between hypoxia andnormoxia (see Figure 3). This was due to the fact that, in hypoxia,the hatchlings thermogenic response was reduced (see “Oldergroup (12–24 hr old)” and “Younger group (�8 hr old)”); there-fore, even though the bath temperature was the same, the Ta withinthe respirometer was less in hypoxia for both age groups.

Older group (12–24 hr old). The thermogenic response toacute cold was a progressive increase in VO2, which, by 30 min,

Figure 1. Time trajectories of ambient temperature during the step-change protocol in normoxia and hypoxia. The two curves did not differsignificantly at any time.

76 AWAM, CATANA, AND MORTOLA

was about 70% higher than in the warm environment (Figure 4,top). In hypoxia, the thermogenic response was less than half thenormoxic response. Tb at the onset of the experiment averaged39.9 � 0.1 °C; in the cold, Tb dropped, significantly more so inhypoxia (34.0 � 0.8 °C) than in normoxia (36.8 � 0.7 °C;one-way repeated measures ANOVA).

Different from VO2, the number of calls reached its peak duringthe first few minutes in the cold, whether in normoxia or hypoxia,and remained constant thereafter, with similar values irrespectiveof the level of oxygenation (Figure 4, second panel from top). Theamplitude and pitch of the calls remained approximately the sameirrespective of the condition (Figure 4, bottom panels).

Younger group (<8 hr old). In this age group, the increasein VO2 in the cold (from about 0.75 to 1 ml/min or �30%; Figure5) was significantly less than in the older hatchlings. This wasexpected owing to their younger age (Szdzuy et al., 2008). Thetime profile of the number of calls (Figure 5, second panel fromtop) indicated a rapid increase and a subsequent decline (Blumberg& Stolba, 1996). However, when analyzed statistically, the numberof calls did not differ significantly from the older group, and thecalls were characterized by slightly higher pitch and lower ampli-tude.

During hypoxia, as in the older group, the thermogenic responseof the young hatchlings was severely blunted; with it, therefore, Tbdecreased markedly (Figure 3). The number of calls was less thanin normoxia only in the early phases of cold; later, hypoxia had nosignificant effects. Hence, as in the older group, by 30 min of cold(Figure 5, right panels), VO2 was much less than in normoxia,while the number of calls and their sound properties remained thesame.

VO2-Calls Relationship

As apparent from figures 4 and 5, both young and older hatch-lings reached the peak of their vocalization before reaching themaximal VO2. Therefore, both in normoxia and hypoxia, the iso-time plot of the changes in VO2 against the changes in number ofcalls invariably originated a loop with a clockwise direction (Fig-ure 6), indicating that the autonomic response lagged behind thecall response.

The Cost of Calls

During the three 5-min bins of the warm condition, on average,the number of calls remained very similar (Figure 4, second panelfrom top). Nevertheless, within each animal, differences did occur.Therefore, it was of interest to consider whether or not, during thewarm condition, interbin differences in number of calls (i.e., thedifferences between each pair of the three 5-min bin; X-axis)

Figure 2. From top to bottom, oxygen consumption (VO2), number ofcalls, and their amplitudes and frequency in hatchlings during step changesin ambient temperature, in normoxia (open symbols), and hypoxia (filledsymbols). Symbols are group means, and bars represent 1 SEM. Withinnormoxia or hypoxia, symbols with same letter are not statistically differ-ent; � � significant difference between normoxia and hypoxia (two-wayrepeated measures ANOVA, p � .05); arrows indicate the average lowercritical temperatures.

Figure 3. Time trajectories of ambient temperature during acute exposureto cold in young (�8 hr old, dashed lines) and older (12–24 hr old,continuous lines) hatchlings during normoxia (open symbols) or hypoxia(filled symbols). � � significant difference between hypoxia and normoxia(two-tailed paired t test, p � .05).

77THERMOGENIC AND VOCALIZATION RESPONSES TO COLD

entailed detectable difference in VO2 (Y-axis). The slope of suchrelationship represents the O2 used (�l) per call. At the youngerage, the slope of the data points in normoxia, hypoxia, or allcombined was 0.7 �l O2/call � 0.8 (STDEV of the slope; N �102), not significantly different from 0 (Figure 7, top panel). At theolder age, the slopes were significantly different from 0, both innormoxia and hypoxia ( p � .001), and were not significantlydifferent from each other. Their values were 2.1 �l O2/call � 0.6(STDEV of the slope; N � 51) in normoxia, 2.4 � 0.8 in hypoxia(N � 51), and 2.2 � 0.5 by combining the normoxic and hypoxicvalues (N � 102; Figure 7, bottom panel).

The product between the number of calls and the O2 cost of eachcall gave the O2 used for the calls; this value was subtracted fromthe total VO2 , and the results represented the VO2 free of the O2

cost of vocalization (dashed lines in the top panels of figures 4 and5). The difference between the continuous and dashed lines is

small; in fact, the O2 cost of vocalization never exceeded 10% ofthe total VO2 during the thermogenic effort.

Discussion

At the onset of the study, we were expecting some degree ofreciprocity between the vocalization and the autonomic responsesto cold and hypoxia, meaning that the reduction or the failure ofthe latter would entail some increase in the former. The results didnot conform to the expectations. In fact, (a) in the early postnatalhours, the vocalization response to cold did not compensate for thelow thermogenic capacity of this age; (b) the vocalization responsedid not compensate for the hypoxic depression of autonomicthermogenesis; and (c) hypoxia had a different effect on vocaliza-tion and the autonomic response to cold; it blunted the latter andleft the former unaltered.

Figure 4. Hatchlings at 12–24 hr of age (average 16.9 hr � 1.2). Left panels. Time trajectories of oxygenconsumption (VO2, top), number of calls, sound amplitude, and frequency during warm and cold conditions (atthe ambient temperature of Figure 3) in normoxia (open circles) and hypoxia (filled triangles). The dashed linesin the top panel indicate VO2 without the cost of calls. Symbols represent group means; bars are 1 SEM; � �significant difference from the corresponding normoxic value (two-tailed paired t test, p � .05).Right panels.Average values during the 15 min of warm (W) and during the last 10 min of cold (C) in normoxia (Nx) orhypoxia (Hx). Columns represent group means; bars are 1 SEM; # � significant difference from the corre-sponding warm; � � significant difference from the corresponding normoxia (two-way repeated measuresANOVA, p � .05).

78 AWAM, CATANA, AND MORTOLA

General and Methodological Considerations

Behavioral means of responding to changes in ambient condi-tions and, in particular, to a cold stimulus, are ancient responsesthat must have evolved much before the autonomic control of Tb.In fact, all classes of animals depend on behavioral mechanismsfor the control of Tb. Important means for endogenous heat pro-duction have evolved only in birds and mammals, and even inthese classes, behavioral responses remain an essential defenseagainst cold (Bicego et al., 2007). In newborn mammals and birds,the large body surface-to-volume ratio and the modest control ofbody heat dissipation are such that heat loss easily overwhelmsheat production. In addition, the autonomic thermogenic ma-chinery, although functional at birth, has very low capacity(Mortola, 2001, 2009). Hence, newborn birds and mammalsrely heavily on behavioral responses to protect Tb during coldby searching warmer places (thermotaxis) to optimize Tb and

metabolic rate (Leonard, 1974; Fowler & Kellogg, 1975; Hull,Hull, & Vinter, 1986; Fewell, Kang, & Eliason, 1997), findingshelters in burrows (Haim, Van Aarde, & Skinner, 1992),assuming a curled or fetal posture (Mortola & Feher, 1998), orby huddling with siblings to limit the exposed body surface(Alberts, 1978; Alberts & Brunjes, 1978). The physical contactwith the parent is probably the most efficient behavioral methodof protection against cold. The interpretation of the neonatalvocalization in the cold, as the pup’s behavioral response con-ceived to attract parental attention, could be incorrect (Blum-berg & Alberts, 1990). Nevertheless, vocalization, from theloud crying of the human infant to the ultrasound production ofsome neonatal rodents (Elwood & Keeling, 1982; Blake, 1992;Blumberg, Efimova, & Alberts, 1992; Blumberg & Stolba,1996), often does attract attention and, in this way, contributesto the protection of the neonate against cold.

Figure 5. Hatchlings �8 hr old (average 4.1 hr � 0.4). Left panels. Time trajectories of oxygen consumption(VO2, top), number of calls, sound amplitude and frequency during warm and cold conditions (at the ambienttemperature of Figure 3), in normoxia (open circles) and hypoxia (filled triangles). The dashed lines in the toppanel indicate VO2 without the cost of calls. Symbols represent group means; bars are 1 SEM; � � significantdifference from the corresponding normoxic value (two-tailed paired t test, p � .05). Right panels. Averagevalues during the 15 min of warm (W) and during the last 10 min of cold (C) in normoxia (Nx) or hypoxia (Hx).Columns represent group means; bars are 1 SEM; # � significant difference from the corresponding warm; � �significant difference from the corresponding normoxia (two-way repeated measures ANOVA, p � .05).

79THERMOGENIC AND VOCALIZATION RESPONSES TO COLD

In adult mammals, the Ta chosen in a temperature gradient, orthermocline, is a common behavioral indicator of the thermoreg-ulatory response to a change in environmental conditions. Innewborns, the lack of full locomotory control complicates theapplication of this approach (Johanson, 1979; Kleitman & Sati-noff, 1982). Huddling can have complex effects on the newborn’s

thermogenic response. For example, in precocial species like thepig or the porcupine, huddling in the cold decreases the newborn’sneeds for thermogenesis and lowers VO2 (Haim et al., 1992;Mount, 1979). Differently, in altricial species like the neonatal rat,the cold-induced drop in Tb lowers VO2 by the Q10 effect; in thiscase, huddling, by protecting Tb, raises VO2 (Saiki & Mortola,1996). Therefore, it was considered preferable to study each hatch-ling individually rather than in a group. In chicken hatchlings,differently from some neonatal mammals, calls are stereotyped(Bugden & Evans, 1991, 1999; Marx et al., 2001), with only minorchanges in sound characteristics (pitch and amplitude), as con-firmed by the results of the present experiments.

We have maintained the experimental conditions, including thecold stimulus of either protocol, as constant as possible betweennormoxic and hypoxic runs so that the level of oxygenation wasthe only independent variable. By alternating the order of exposureto normoxia and hypoxia, we should have eliminated the possibil-ity of a carry-over effect of the first into the second exposure andthe possibility of time-dependent habituation to the experimentalconditions.

Correlation Between Vocalization and Thermogenesis

Several considerations lead to the expectation of some degree ofreciprocity between autonomic thermogenesis and behavioralmeans of Tb control (Weiss& Laties, 1961; Myhre, 1978; Bugdenand Evans, 1997; Schlader et al., 2009). For example, lowervertebrates with minimal endothermy rely heavily on behavioralmeans for Tb regulation. Ontogenetically, the preferred Ta grad-ually declines as endogenous thermogenesis develops (Hull &Hull, 1982; Hull et al., 1986). In rat pups, calls in the cold areminimal, as long as the thermogenic effort suffices, and increasewhen the cold stimulus overwhelms the thermogenic effort (Blum-berg & Stolba, 1996; Sokoloff & Blumberg, 1997). Neonates withless thermogenesis show greater thermotaxis (Leonard, 1982).

On these grounds, we were expecting some reciprocity betweenvocalization and thermogenesis in the control of Tb, but the resultsproved otherwise. In fact, in the early postnatal hours, when

Figure 6. Oxygen consumption (VO2) against the number of calls during cold, expressed in percent of theaverage value in warm conditions. All loops had a clockwise direction (arrows), indicating that the behavioralresponse (increase in number of calls) anticipated the autonomic response (increase in VO2).

Figure 7. Difference in number of calls between each pair of the three5-min periods in the warm condition, plotted against the correspondingdifferences in oxygen consumption (VO2, ml/min). Continuous line is thebest-fit linear regression; dashed lines are the 95% confidence intervals.The slope of the line represents the VO2 of one call.

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autonomic thermogenesis is less than in the following hours (Szd-zuy et al., 2008), the number of calls was not significantly higherthan at the older age. Equally, in hypoxia, when thermogenesis wasseverely blunted, the number of calls was not greater than innormoxia (see Figure 4 and Figure 5). One possibility is that thehatchling’s calls have an all-or-none pattern, meaning that theirnumber does not bear any proportionality to the magnitude of thestimulus. This agrees with the finding that the calls did not increasein number as the cold exposure progressed with time (see Figure 4and Figure 5) or intensity (see Figure 2). In warm conditions,during hypoxia, both age groups increased the pitch of their calls(see figures 2, 4, and 5, bottom panels), and the same occurred inthe younger hatchlings during normoxic cold. In this regard, it is ofinterest that some adult songbirds confronted by a loud ambientnoise make themselves heard by switching to high-pitched sounds(Slabbekoorn & Peet, 2003; Wood & Yezerinac, 2006; Brumm,2006), probably because the increase in pitch is more effective forrecognition than the increase in call amplitude or pulse rate.Hence, one may argue that a change in sound characteristics, ratherthan in the number of calls, was the vocalization pattern adoptedby the hatchlings to signal the situation of stress during cold orhypoxia. However, when cold and hypoxia occurred simultane-ously, neither the number nor the sound characteristics of the callsdiffered significantly from cold normoxia. Therefore, we concludethat there is no consistent relationship between vocalization andthermogenesis.

We estimated the cost of each call at �0.7 �l of O2 in theyounger group and �2.2 �l of O2 in the older one. Perhaps this agedifference may be related to the higher loudness (greater ampli-tude) of the calls of the older group. In either case, however, thecost of calls was extremely small. In the American white pelicanembryos at the pipping stage, the cost of calls at rest was computedat �0.02 �l/g (Abraham & Evans, 1999); the embryo’s weight wasnot reported, but if we assume that it is around 100g (Pearson,Seymour, Baudinette, & Runciman, 2002), the cost of each call inthe pelican embryo would be almost identical to what we measuredin the older chicken hatchlings. From the number of calls and theircost, we estimated that the vocalization during cold contributed, atthe most, 10% of the thermogenic effort (Figure 4 and Figure 5).Previously, McCarty (1996), in the nestlings of five out of sevenavian species, was unable to demonstrate a significant increase inVO2 during begging; in the remaining two species, the VO2 increasewas 5% and 27%. It can be concluded, therefore, that vocalizationis a very economical activity, which justifies its major role amongthe responses to cold and its immediate recruitment before the fullmanifestation of autonomic thermogenesis (see Figure 7).

Effects of Hypoxia on Vocalization and Thermogenesis

Hypoxia blunts the thermogenic response to cold, and avianhatchlings are no exception (Mortola, 2009). Some behavioralresponses, like the choice of a Ta that minimizes energy expendi-ture and favors heat loss, and the lesser propensity to huddle, havecontributed to the idea that hypoxia shifts the set point for thecontrol of Tb to a lower value (Wood, 1991; Gordon & Fogelson,1991; Dupre & Owen, 1992; Tattersall & Milsom, 2003; Scott etal., 2008; Atchley, Foster, & Bavis, 2008). Autonomic and behav-ioral responses to hypoxia probably are coordinated centrally(Pereira et al., 2006; Guerra et al., 2008). The current findings of

the drop in VO2 sensitivity to cold and in the critical Ta are inagreement with the concept of a hypoxic decrease in the thermo-regulatory set point.

In contrast, hypoxia did not alter the vocalization response tocold in any aspect, number, amplitude, and pitch of the calls. Oneinterpretation could be that the hatchling’s calls are not strictlybehavioral responses. The view that calls are simply acousticphenomena associated with breathing was proposed by Blumbergand Alberts (1990) from experiments on neonatal rats. Theseauthors suggested that the ultrasound vocalization of the pupsduring cold was an acoustic by-product of laryngeal brakingthrough narrowing of the vocal folds, a phenomenon commonlyobserved in neonatal mammals to raise airway pressure and im-prove lung expansion (Mortola, 2001). According to this view(Blumberg & Alberts, 1990, 1991), the higher the metabolic de-mands and pulmonary ventilation, the greater the frequency ofbreaths with laryngeal braking and ultrasounds. However, otherexperiments in neonatal rats and mice have demonstrated thatthermogenesis and ultrasound calls can occur independently andare not physiologically linked (Hofer & Shair, 1991; Bollen et al.,2009). In avian hatchlings, expiratory braking has never beenobserved. In addition, the loops in Figure 7 do not support the ideaof a proportionality between VO2 and calls. Hence, it seems likelythat vocalization is an important response to cold but is notphysiologically linked to thermogenesis; as a consequence, inhypoxia, vocalization would be excluded from the general strategyof downregulation of thermogenesis and behavioral responses tocold. The evolutionary advantage of the mismatch between vocal-ization and the thermogenic response to cold is hypothetical.Because calls require respiratory airflow, the nonproportionalitybetween vocalization and thermogenesis should limit the respira-tory evaporative heat loss and the risk of hypothermia. In the eventof hypoxia, the persistence of the call response to cold, despite thedrop in thermogenesis, permits the hatchling to continue signalingits abnormal condition. Because the cost of the calls is minimal,vocalization does not hinder hypoxic hypometabolism. Presum-ably, therefore, the fact that the increased vocalization in responseto cold is not linked to thermogenesis, and is immune from thedepressant effects of hypoxia, represents a safety mechanism thatmay carry some advantage.

Conclusions

The current results support and extend to the avian hatchlingsthe notion that hypoxia lowers thermogenesis, possibly with adecrease in the set point for thermoregulation. More important, theresults show that vocalization is not proportional to the coldstimulus, is unrelated to the degree of autonomic thermogenesis,and is unaltered by hypoxia. This suggests that vocalization in thecold is a stereotyped response not necessarily related to the auto-nomic regulation of Tb. This dissociation could carry some ad-vantage in conditions of cold and hypoxia.

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Received June 21, 2010Revision received October 4, 2010

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83THERMOGENIC AND VOCALIZATION RESPONSES TO COLD