J Comp Physiol A (1989) 165 : 15-26 Journal of

Sensory, Comparative Neural, and

Physiology A .e~av,or~, Physiology

�9 Sprinoer-Verlag 1989

Airflow and pressure during canary song: direct evidence for mini-breaths

Rebecca Schurr Hartley 1 and Roderick A. Suthers 2 1, 2 Department of Biology and 2 School of Medicine, Indiana University, Bloomington, Indiana 47405, USA

Accepted January 11, 1989

Summary. Male canaries (Serinus canaria) produce songs of long duration compared to the normal respiratory cycle. Each phrase in a song contains repetitions of a particular song syllable, with repe- tition rates for different syllables ranging from 3 to 35 notes/s. We measured tracheal airflow and air sac pressure in order to investigate respiratory dynamics during song.

Song syllables (11-280 ms) are always accom- panied by expiratory tracheal airflow. The silent intervals (15-90 ms) between successive syllables are accompanied by inspiration, except for a few phrases where airflow ceases instead of reversing. Thus, the mini-breath respiratory pattern is used most often by the five birds studied and pulsatile expiration is used only occasiona]ily.

Songs and phrases accompanied by mini- breaths were of longer duration than those accom- panied by pulsatile expiration, presumably because the animal's finite vital capacity is not a limiting factor when the 'volume of air expired for one note is replaced by inspiration prior to the next. Pulsa- tile expiration was used for only a few syllable types from one bird that were produced at higher repetition rates than syllables accompanied by mini-breaths. We suggest that male canaries switch to pulsatile expiration only when the syllable repe- tition rate is toe high (greater than about 30 Hz) for them to achieve mini-breaths.

Changes in syringeal configuration that may accompany song are discussed, based on the as- sumption that changes in the ratio of subsyringeal (air sac) pressure to tracheal flow rate reflect chan- ges in syringeal resistance.

Introduction

Vocal communication in birds is used primarily for mate attraction and territorial defense (see re-

view by Catchpole 1982) and, as in other verte- brates, these vocalizations are powered by the re- spiratory system. Thus, sound production requires not only that the syringeal muscles move the vocal organ, or syrinx, into a vocalizing configuration, but also that the respiratory muscles generate an expiratory airstream at an appropriate flow rate (Gaunt and Gaunt 1977). Consequently, complex and extended vocalizations such as oscine (song- bird) songs require specific temporal patterns of airflow through the vocal tract. Clearly, therefore, the respiratory pattern that accompanies a song is an integral part of the mechanism of song pro- duction, yet only one previous study has examined respiration in a singing bird - in the canary, Serinus canaria (Calder 1970).

Male canaries sing for up to 25 s, more than 30 times the length of the normal respiratory cycle. An obvious question arises as to how they can achieve such extended song with a limited vital capacity. Their trilled song consists of single notes or syllables usually repeated at high repetition rates (15 to 30/s) with brief silent intervals (20 to 35 ms) between successive notes. Most songs consist of a succession of phrases, each phrase made up of repetitions of a particular syllable (Nottebohm and Nottebohm 1976).

To study respiration during canary song, Calder (1970) used an impedance pneumograph to measure changes in the dorso-ventral dimension of the thorax of singing males. He found that the notes of a sustained trill were synchronized one-to- one with small thoracic oscillations. Moreover, long duration trills were produced with very little overall dorso-ventral compression. These results led him to postulate that male canaries use a 'mini- breath' respiratory pattern during song. He hy- pothesized that each note of the trill is accompa- nied by a brief expiration and each silent interval is accompanied by a brief inspiration. Depending

16 R.S. Hartley and R.A. Suthers: Respiratory dynamics in a singing bird

o n the v o l u m e s of air i n sp i r ed a n d expired, a b i rd us ing the m i n i - b r e a t h p a t t e r n cou ld have a n effec- t ively u n l i m i t e d supp ly of air for song p r o d u c t i o n .

Howeve r , C a l d e r ' s hypo thes i s needs experi-

m e n t a l ve r i f i ca t ion because it is based o n the as- s u m p t i o n s t ha t (1) the d o r s o - v e n t r a l d i m e n s i o n o f the t h o r a x accu ra t e ly ind ica tes the p ressure level w i th in the r e sp i r a to ry sys tem a n d (2) b o t h syr inx a n d glot t is o p e n d u r i n g the b r i e f pause b e t w e e n successive tri l l no tes to a l low i n s p i r a t o r y airf low. G a u n t et al. (1973) d i sagreed wi th Ca lde r ' s in te r - p r e t a t i o n o f his d a t a a n d sugges ted in s t ead t ha t the thorac ic osc i l l a t ions obse rved by C a l d e r re- sul ted f r o m pu l sed exp i ra t ion . T h e y a r g u e d tha t the r ap id o p e n i n g a n d c los ing of a syr ingea l va lve wi th tho rac i c c o m p r e s s i o n at a g iven level w o u l d p r o d u c e such m o v e m e n t s . As G a u n t a n d G a u n t (1985a) p o i n t e d out , the cr i t ical ev idence for a n i n s p i r a t o r y phase w o u l d be m e a s u r e m e n t o f nega- tive i n t e r n a l pressure . These a u t h o r s sugges ted t ha t a ' h i g h - r e s i s t a n c e syr inx t ha t ext rac ts a m a x i m u m a m o u n t o f s o u n d f r o m a m i n i m u m a i r f l o w ' w o u l d a l low b i rds to s ing for l o n g per iods u s ing a l imi t ed

air supply . I n this paper , we p r e sen t the first d i rect m e a -

s u r e m e n t s o f t r achea l a i r f low a n d air sac p ressure in a s ing ing b i rd . O u r resul ts show tha t ma le c a n a r - ies use the m i n i - b r e a t h r e sp i r a to ry p a t t e r n m o s t o f the t ime, a l t h o u g h we did observe the pu lsa t i l e e x p i r a t i o n p a t t e r n in a few cases.

Materials and methods

Adult male canaries of the Wasserschlager strain, with a mean weight of about 22 g, were maintained on a normal, seasonally changing photoperiod; experiments were performed from May to August during their natural song season. A few weeks prior to an experiment a 10-mg pellet of testosterone propionate (In- novative Research of America) was implanted subcutaneously to promote song and thus increase the chance of the bird singing while instrumented. Males were housed in small cages within which they moved freely both prior to and during experiments. Pre-operative song was recorded using a Panasonic dynamic microphone (WM-2298), mounted 15-35 cm from the bird, with a cassette recorder (Marantz PMD221). The frequency response of this system was _+3 dB from 500 Hz to 4.5 kHz (and _ 6 dB up to 11 kHz).

During all surgeries to implant flow and pressure transduc- ers, the birds were anesthetized with halothane (Fluothane, Ayerst). Atropine sulfate (1.4 gg, Lilly) was administered prior to tracheal flow probe insertion to reduce airway secretions. Birds that sang during experiments typically did so 1-3 days after surgery.

Anterior thoracic air sac pressure was measured with a temperature-compensated miniature piezoresistive pressure transducer (Endevco model 8507-5 or 8507B-5), having a re- sponse within ___3 dB from DC to 13 kHz and a resonant frequency of 65 kHz. The transducer was attached with a piece of self-gripping fastener (Velcro) to an elastic belt worn by

g,ott,s I F trachea

syrinx bronchus

/ ~ ! r

a i r sacs I I \

Fig. 1. Schematic anatomical diagram showing positions of the tracheal thermistor bead (F) and the air sac pressure cannula (P). (After Suthers and Hector 1982)

the bird and was positioned on his back, with the associated leads routed out of the top of the cage. The transducer was connected to the air sac lumen via a 4-cm length of size 18 (ID 1.07 mm) polyvinylchloride tubing (Alpha), which was in- serted through the ventral body wall (Fig. 1) and secured with sutures and tissue adhesive (Histoacryl blue, B. Braun Melsun- gen, Federal Republic of Germany). The output voltage was recorded in the FM mode on one channel of an instrumentation tape recorder (Racal 4DS). Data were recorded at a tape speed of 3-3/4 ips (9.5 cm/s), giving a frequency response of DC to 2.5 kHz for this pressure recording system. Subsyringeal (bron- chial) pressure was assumed to be equal to anterior thoracic air sac pressure (Brackenbury 1972).

The flow transducer consisted of a microbead thermistor (Thermometrics B07JA202N or B07KA202N), nominally 7 mil (0.18 mm) in diameter, mounted on the end of an epoxy-coated wire support. The bead was positioned in the center of the tracheal lumen by inserting it through a small hole in the ventral side of the trachea (Fig. 1) and attaching its wire support to the outside of the trachea with tissue adhesive. Fine leads from the flow probe were routed under the skin to the animal's back and soldered to gold pin connectors fastened on the elastic belt. Heavier leads then exited out of the top of the cage. The thermistor was heated and maintained at a constant tempera- ture by a feedback circuit (designed by C.D. Mills, Duke Uni- versity). The voltage required for the current to heat the therm- istor, which was proportional to the airflow rate past the bead, was tape recorded in the FM mode (frequency response DC to 2.5 kHz). The effects of temperature and humidity inside the trachea were empirically corrected by manually zeroing the circuit after the transducer was in position.

Birds were sacrificed at the end of the experiment in order to calibrate in situ the voltage output of the flow meter circuit against the rate of expiratory airflow through the trachea. Being careful not to disturb the tracheal flow probe, we transected the trachea as far posterior as possible and securely fitted a piece of tubing to the anterior cut end. Humidified air was blown through a tube past a previously calibrated thermistor and into the posterior end of the bird's trachea. The bird's beak was propped open so air could flow freely out of the glottis. As the airstream was varied slowly over a wide range of flow rates, we recorded the output voltages for both therm-

R.S. Hartley and R.A. Suthers: Respiratory dynamics in a singing bird 17

Fig. 2. Rate of tracheal airflow (F) and anterior thoracic air sac pressure (P) during quiet respiration in Bird 1. Flow reversals are marked by arrows. i inspiration; e expiration

istors. We assumed that both transducers experienced the same flow rate at any instant and that the tracheal thermistor's re- sponse to inspiratory vs. expiratory airflow was the same. Since the relationship between voltage output and airflow rate was non-linear, the flow signal recorded during experiments was later linearized and integrated as described by Suthers and Hec- tor (1982) to obtain the rate of airflow and breath volumes.

For three of our five successful experiments (Birds 3-5) we used a two-bead directional flow probe, similar to those employed by Suthers and Hector (1982) and Bernstein and Schmidt-Nielsen (1974). With these probes we verified that ex- piration is always accompanied by positive air sac pressure and that inspiration is always accompanied by negative pressure, relative to atmospheric pressure. In the other two experiments (Birds 1 and 2) we used a single-bead, non-directional flow probe and relied on air sac pressure data to determine the direc- tion of airflow.

During experiments the Panasonic microphone output was amplified (Princeton Applied Research, model 113 pre-amp) and recorded in the direct mode on a third channel of the tape recorder. This sound recording system had the same fre- quency response as the one used for pre-op recordings. Spectro- grams were made of both pre- and postoperative song (Un- iscan II, Multigon).

Casts of the trachea, bronchi, lungs, and air sacs were made from dead birds by injecting silicone rubber sealant (G.E. 4~ 54W03) down the trachea. The tissues were later macerated in KOH so that the casts could be recovered intact. Dead space volumes were estimated from these casts by measuring their water displacement.

Results

Silent respiration

Respi ra tory rates in resting, non-vocal izing canar- ies are between 1.3 and 1.8 Hz. Peak expiratory tracheal flow rates during quiet respiration range f rom 0.7 to 3.6 ml/s, whereas peak expiratory air sac pressures are abou t +0 .5 to +3 .0 cm H 2 0 . Peak inspiratory flow rates are between 1.4 and 3.5 ml/s; peak inspiratory pressures are a round - 0 . 5 to - 2 . 5 cm H 2 0 . In our example o f quiet respiration, the respiratory rate and peak flow and pressure values are slightly higher than those men- t ioned above because this segment occurred shortly before the start o f a song (Fig. 2). Note that the flow trace shows a positive deflection regardless of airflow direction. We therefore used the pressure

Table 1. Respiratory rates and tidal volumes during quiet respi- ration

Bird Respiratory rate Mean tidal volume ~ no. (Hz)

(ml) (n)

1 1.7 (quiet respiration) 0.54_+0.10 26 2 1.8 (quiet respiration) 0.31 _+ 0.06 24

1 2.0 (before 25-s s o n g ) 0.64_+0.16 16 I 3.3 (after 25-s song) 1.23___0.34 16

a Inspired and expired volumes were estimated separately and then averaged. Mean +_ standard deviation

data to determine the direction o f flow, as de- scribed above. Birds I and 2 had somewhat differ- ent mean tidal volumes (Table 1). The combined dead space of the trachea and pr imary bronchi of the male canary was estimated at 0.09 _+ 0.01 ml (n=3).

Typically, respiratory rates increase prior to the onset o f song and are high following a song. Peak tracheal flow rates are also higher at these times than in the resting bird. We quantified these chan- ges for the eight respiratory cycles that immediate- ly preceded a 25-s song and for the eight cycles that immediately followed the same song (Bird 1). Our choice o f eight rather than some other number of cycles was arbi t rary as the changes were most ly gradual. Dur ing the eight cycles before song the mean respiratory rate was 2.0 Hz ; after song the rate was 3.3 Hz. Prior to song the peak inspiratory flow rate rose f rom 3.7 to 7.3 ml/s with mos t o f the increase occurring on the last cycle. After song the peak inspiratory flow rate d ropped f rom 12.4 to 8.0 ml/s. The mean tidal volume for the 8 cycles preceding the song was somewhat higher than at rest and the mean volume following the song was more than twice the resting value (Table 1).

Respiration during song

The songs produced during experiments appeared to be normal. In only a few cases the successive

18 R.S. Hartley and R.A. Suthers: Respiratory dynamics in a singing bird

Fig. 3a, b. Long 'whistled' notes from Bird 2. a Spectrogram shows the last part of a 3.7-s song. Segment between arrows is shown in b. b Vocalizations (S), rate of tracheal airflow (F), and air sac pressure (P). The combination of high tracheal flow rate and negative air sac pressure, which occurs during the pauses between notes, clearly indicates an inspiratory phase. Inspiration-to-expiration flow reversals are marked by diagonal pointers. Note repetition rate is 6.5 Hz

repetitions of a particular syllable were slightly more variable in sound frequency structure during an experiment than they were pre-operatively. The longest song recorded from each of five birds dur- ing an experiment lasted between 7 and 25 s. The repetition rates for different syllables ranged from 2 to 36/s with syllable durations between 11 and 280 ms and silent interval durations between 15 and 90 ms.

Tracheal airflow and anterior thoracic air sac pressure were recorded simultaneously during song production in 5 male canaries; the respiratory pat- terns that accompany song were determined for all five. However, we successfully calibrated tra- cheal flow rates in only two individuals (Birds 1 and 2). The assortment of 6 syllable types described below was selected from these two birds to repre- sent the range of respiratory patterns observed.

The long 'whistled' syllables of Bird 2 were produced during expiration, as were all sounds we recorded (Fig. 3). The pause between notes was accompanied by both a negative pressure in the

air sac and also high airflow through the trachea as compared to quiet respiration. This combina- tion of pressure and flow clearly indicates inspira- tion. Thus, during this phrase, one expiration-in- spiration cycle accompanied every syllable. The flow trace does not drop to zero during airflow reversals because the transient response of the flow transducer was not fast enough to follow such ra- pid changes, and so the inspiration-to-expiration reversal is indicated only by a small inflection (Fig. 3 b). Prior to sound onset the tracheal flow rate decreased at the same time as the subsyringeal pressure was increasing. Thus, each syllable was probably initiated by the bird increasing the resis- tance of the syrinx. Flow and pressure increased slightly during each syllable. Sound production continued until the subsyringeal pressure had dropped almost to zero. The rapid onset of inspira- tory airflow after each syllable suggests that the syringeal resistance was immediately decreased. Low syringeal resistance during the inspiratory phase is also implied by the relatively high peak flow rate and its accompanying low amplitude neg- ative pressure (Fig. 3 b). The volumes of air expired and inspired for each syllable and silent interval, respectively, were closely matched (Table 2). Inter- estingly, after the last syllable of this phrase the bird inspired once and then did not breathe for 0.6 s. We recorded zero flow and zero pressure dur- ing that period. This brief cessation of breathing following song was not common; we observed it

R.S. Hartley and R.A. Suthers: Respiratory dynamics in a singing bird 19

Fig. 4a-e. Complex syllable from Bird 2. a Spectrogram of a 1.2-s isolated phrase. Segment marked by bracket is shown in b. Segment between arrows is shown in c. b Enlarged spectro- gram displaying two elements per syllable, e Sound, tracheal airflow, and air sac pressure. Both elements of each syllable were produced during expiration. After each song element air- flow drops to zero while pressure remains positive, suggesting that the syrinx closes. Inspiration occurs between consecutive syllables. Syllable repetition rate 10.2 Hz

a few times in this same bird and once in another male.

A complex syllable produced by Bird 2 is shown with its corresponding respiratory data in Fig. 4. Both parts or elements of each syllable were produced during expiration. During the brief silent period of about 11 ms between the two elements comprising each syllable, air sac pressure remained positive while tracheal airflow ceased. This combi- nation of positive pressure and zero flow suggests that the syringeal lumen was completely occluded. The silent intervals between syllables were accom- panied by inspiration. Although the total volume of air expired per syllable appears to be slightly greater than the volume of air inspired between syllables, the differential is within our margins of error and hence is probably not significant (Ta- ble 2).

The mini-breath respiratory pattern was used

Table 2. Repetition rate, duration, and volume of air expired and inspired, for selected syllables of canary song a

Fig- Bird Syl- Syllable Expi- Mean volume ure no. lable duration ration per note b no. repe- (ms) or

tition inspi- (ml) (n) rate rat ion (Hz)

3 2 6.5 119 E 0.25 +0.02 6 I 0.26 __0.02 6

4 2 10.2 38 (part 1) E 0.07 • 9 14 (part 2) E 0.005_+0.002 9

I 0.06 _+0.02 9

5 1 21.6 16 E 0.12 • 20 I 0.11 +0.02 20

6 1 29.9 11 E 0.04 +0.01 15 I 0.05 +0.01 15

7 2 32.1 I1 E 0.007+0.002 29 I none

8 2 18.8 21 (part 1) E 0.02 • 10 10 (part 2) E 0.003__.0.001 10

I none

" Data correspond to the phrases listed in the same order b Mean_+ standard deviation

shown in figures and are

also by Bird 1 for the two phrases with high repeti- tion rates shown in Figs. 5 and 6. As in Fig. 3 the flow trace does not reach zero during flow re- versals because of the transducer's transient re-

20 R.S. Hartley and R.A. Suthers: Respiratory dynamics in a singing bird

Fig. 5a, b. Fast trill (21.6 Hz) from Bird 1. a Spectrogram from the middle of a 13.4-s song. Segment between arrows is part of a 5.5-s phrase, b Sound, tracheal airflow, and air sac pressure. Prior to sound onset the expiratory flow drops to zero even though air sac pressure remains positive, suggesting closure of the syrinx. Inspiration-to-expiration flow reversals marked by diagonal pointers

sponse limitations. In both phrases expiratory flow dropped to zero prior to the onset of sound (Figs. 5 and 6). The accompanying positive air sac pressure suggests that the syringeal lumen was completely occluded prior to sound initiation. During syllable production the air sac pressure dropped as the tra- cheal flow rate increased; this combination sug- gests a decreasing syringeal resistance. Syllables ended when the airflow reversed. The volumes of air inspired and expired during such rapid cycles were similar to each other and were small com- pared to the normal tidal volume (Table 2).

A fundamentally different respiratory pattern was used for 3 of the 22 syllable types recorded from Bird 2. Of 96 phrases examined, 13 were ac- companied by pulsatile expiration instead of mini- breaths. The most common of these three syllable types (used in 11 of the 13 pulsatile expiration phrases) was produced at a rate of 32.1-35.0/s (Fig. 7). The other two syllable types each occurred only once in 96 phrases. Although they had repeti- tion rates much lower than the most common sylla-

ble (18.8 and 15.8 Hz), the rates were effectively twice that because each syllable contained two ele- ments that were produced in separate expiratory pulses (Fig. 8). In each of these cases, air sac pres- sure remained positive throughout the entire phrase, indicating all airflow was expiratory. The drop of flow rate to zero between successive notes suggests that the syringeal lumen was completely occluded during the silent interval. The peak flow rate was relatively low while air sac pressure re- mained fairly high (Figs. 7 and 8). The volume of air expired for each syllable was small (Table 2). The 11 phrases containing the syllable type shown in Fig. 7 ranged from 0.5 to 1.1 s in duration. They usually appeared alone, but occasionally were fol- lowed after a short pause by a mini-breath phrase. The third syllable type, which is not shown in a figure here, was 0.4 s in duration and came at the end of a mini-breath song.

In several instances we recorded respiratory data patterned like those that accompany song, but the bird did not produce any sound. These 'silent trills' were observed in 3 of 5 males and tended to be of short duration (0.3-0.8 s). Note the similarity between the shapes of the flow and pressure traces for 'silent song' (Fig. 9) and nor- mal audible song (Fig. 3) from Bird 2.

Nearly all syllable types sung by the five male canaries tested were accompanied by the mini- breath respiratory pattern (Table 3). Single-phrase

R.S. Hartley and R.A. Suthers : Respiratory dynamics in a singing bird 21

Fig. 6a, b. Fast trill (29.9 Hz) from Bird 1. a This 1.9-s phrase occurred alone, b Notat ion is the same as in previous figures. Even at this high repetition rate the bird used the mini-breath respiratory pattern

songs as short as 1.0 s showed this pattern. Occa- sionally the first or last few syllables of a song were produced by a continuous expiratory stream which varied in amplitude. The pulsatile expiration pattern was used only for the three syllable types from Bird 2 mentioned above.

Discussion

Mechanism of song production

In songbirds the syrinx is located at the tracheo- bronchial junction (Fig. 1). Two structures located in the anterior end of each bronchus, the medially positioned internal tympaniform membrane and the laterally positioned external labium, are thought to be involved in sound generation (see reviews by Brackenbury 1982; Gaunt and Gaunt 1985a). The precise mechanism of sound genera- tion is not known, but it is generally agreed that several pairs of syringeal muscles control the con-

Table 3. Summary of songs examined, including number of syllables accompanied by mini-breaths or pulsatile expiration, for 5 male canaries

Bird Total song Total no. Durat ion No. of syllable Respiratory No. of Syllable no. duration of songs of single songs types/song pattern syllable repetition rates

(s) (s) types (Hz)

1 347 31 1.5-24.6 1-6 mini-breath 9 4.1-29.9 puls. expir. 0 -

2 94 46 0.5-7.0 1-6 mini-breath 19 3.0-25.0 puls. expir. 3 31.6-37.6

3 142 27 1.8-i 3.3 1-8 mini-breath 11 2.4-30.5 puls. expir. 0 -

4 22 6 1.3-6.7 1-6 mini-breath 11 2.4-27.8 puls. expir. 0 -

5 49 8 3.4-10.2 3-8 mini-breath 12 7.8-28.3 puls. expir. 0 -

22 R.S. Hartley and R.A. Suthers: Respiratory dynamics in a singing bird

Fig. 7a, b. Fast trill (32.1 Hz) accompanied by pulsatile expira- tion, from Bird 2. a Phrase containing arrows lasted 1.1 s and was followed by a 4.4-s song. b Tracheal flow is all expiratory. Between consecutive syllables the syrinx closed, as can be seen from the combination of zero flow and positive pressure

figuration of the vocal organ and that when the membranes and labia are in an appropriate posi- tion they will interact with the expiratory airstream to generate sound. Both syringeal and respiratory muscles must therefore be coordinated for normal song production.

We are able to draw some general conclusions regarding syringeal configuration if we assume that changes in the quotient of subsyringeal (air sac) pressure over tracheal flow rate reflect changes in syringeal resistance, and we postulate that adduc- tion or abduction of the syringeal membranes is the means by which syringeal resistance is in- creased or decreased, respectively. As we measured airflow in the trachea and not in the right and left bronchi, we cannot compare the behavior of the right and left sides of the syrinx (Not tebohm and Not tebohm 1976; McCasland 1987). For the following discussion we assume that the glottis re- mains open during song production. This assump- tion is supported by the fact that peak tracheal pressures during song were (1) low compared to peak air sac pressures during song and (2) similar to peak tracheal pressures during quiet respiration

when the glottis must remain open (Hartley and Suthers, unpublished data).

Our mini-breath results suggest that the syr- ingeal membranes are abducted during the inspira- tory port ion of the mini-breath cycle. During voca- lization it appears that the membranes are partially adducted to a phonatory position. Our data show- ing that syringeal resistance changes during the ex- piratory port ion of the cycle, either at the time of sound onset (Figs. 5, 6, and the second element in Fig. 4) or during individual syllables (Figs. 5 and 6), suggest that the syringeal muscles actively vary the syringeal aperture during the mini-exhalation. In most cases syllables were terminated as both the expiratory flow rate and air sac pressure dropped to zero just prior to flow reversal (Figs. 3, 5, and 6), and it is unclear what the syringeal con- figuration might be. Sometimes it appears that complete syringeal adduction accompanied sound termination (Fig. 4). The rapid expiration-inspira- tion cycles accompanying mini-breath trills are presumably generated by the respiratory muscles. Thus, every syllable in a phrase appears to be the product of precisely coordinated motor outputs to both syringeal and respiratory muscles.

In the case of the pulsatile expiration pattern, the expiratory muscles could have produced a con- stant level of compression while the syringeal mus- cles alone may have been responsible for changes in flow rate as in Gaunt et al.'s (1976) 'oscillating valve' model. Alternatively, as in their 'pulsatile

R.S. Hartley and R.A. Suthers: Respiratory dynamics in a singing bird 23

Fig. 8a-e. Complex syllable accompanied by pulsatile expira- tion, from Bird 2. a This 0.5-s phrase occurred alone. Segment marked by bracket is shown in b. Segment between arrows is shown in c. b Enlarged spectrogram displaying two elements per syllable, e During song all airflow is expiratory. Each ele- ment is produced with a separate pulse of air. Syllable repetition rate 18.8 Hz

input ' model, flow modulation could have been achieved by changes in expiratory pressure, while the syrinx remained fixed in a phonatory position. Our data suggest the birds use a combination of these two mechanisms. Because tracheal airflow varied back and forth from 0 ml/s to 1-2 ml/s while air sac pressure remained positive, it appears that the syringeal membranes were alternately com- pletely adducted and then partially abducted. Be- cause air sac pressure increased when tracheal flow was zero and also air sac pressure decreased as flow decreased, it seems that the level of compres- sion was increased and then decreased with each syllable. As in the case of the mini-breath pattern, we find that both syringeal and respiratory muscles are actively participating in song production. We hypothesize that male canaries switch to pulsatile expiration only when the rate of' note production becomes too high for them to achieve the ampli- tude of thoracic movements needed for mini- breaths (i.e., above about 30 notes/s; see Table 3).

It is interesting that canaries appear to execute the motor patterns of song without vocalizing

(Fig. 9). The general shapes of the flow and pres- sure traces that accompanied 'silent song' are fairly normal, although the air sac pressure is a bit lower than that which usually accompanies vo- calization. We suggest that this reduced driving force, or perhaps some subtle difference in the con- figuration of the syrinx, was responsible for the lack of sound. Gaunt and Gaunt (1985 b) recorded 'false calls' in the monk parakeet (Myiopsitta mon- achus). Their data were syringeal muscle EMG's and they recorded some 'complete EMG perfor- mances with no resulting sounds'. Although our recording system detected no sounds, the bird may have been able to hear himself. We speculate that silent song may make it possible for birds to re- hearse the motor patterns of song production with- out any audible output.

Utilization of mini-breaths

In these first direct measurements of respiration during bird song, we found that in all but a few phrases, canary syllables are produced during ra- pid expiration-inspiration cycles. Because the vol- ume of air expired per note is the same as the volume inspired between successive notes, males are able to sing for relatively long periods without running out of air. Even though mini-breath vol- umes (Table 2) were sometimes smaller than tra- cheal dead space (0.09 ml), it is possible that nor- mal gas exchange occurred. Normal levels of arte-

24 R.S. Hartley and R.A. Suthers: Respiratory dynamics in a singing bird

Fig. 9a, b. 'Silent trill' (8.9 Hz) following trill with pulsatile expiration, from Bird 2. a This 1.3-s 'phrase' appeared between two audible phrases, b Notation is the same as in previous figures

rial Po2, Pco2 and pH have been measured in me- chanically ventilated pigeons (Columba livia) when tidal volume was only 60% of the dead space vol- ume and the ventilation frequency was high (20 Hz) (Bech et al. 1988). However, the relatively large tidal volumes that usually followed a song suggested that our birds may have become hypoxic during song production and that song duration may be limited by the composition of gas in the respiratory system. After a phrase when the vol- umes inspired and expired were similar to normal tidal volumes (Fig. 3), breathing stopped for a brief period, perhaps because of CO2 washout.

The rates of mini-breath respiration were low enough for complete contraction-relaxation cycles to occur in both inspiratory and expiratory muscles (Calder 1970). Some authors have speculated that the syllable production rate might match the reso- nant frequency of the respiratory system (Calder 1970; Brackenbury 1978a; Gaunt and Gaunt 1985a). If that were the case, then birds might be able to alter the system's resonant frequency to match the note repetition rates for different phrases by changing posture and muscle tone. However, experiments to test whether panting in

heat-stressed birds occurs at the resonant fre- quency of their respiratory systems have not pro- duced clear results (see review by Barnas and Rau- tenberg 1987).

The phrases accompanied by pulsatile expira- tion consisted of short duration notes (11-21 ms) produced at high repetition rates (31.6-37.6 Hz). Production of these brief sounds required only a small volume of air per note (Table 2). Even so, phrases that were accompanied by pulsatile expira- tion were never longer than 1.1 s. Using mini- breaths the same bird sang as long as 7.0 s. Thus, it seems that by providing an effectively unlimited air supply, the mini-breath respiratory pattern al- lows mate canaries to sing for extended periods. Long duration songs may provide a greater stimu- lus to conspecifics and thereby make the singer more attractive to females or more threatening to rival males. They may also make it easier for indi- viduals in a population to develop their own dis- tinct songs.

Comparison of flow rate and pressure values with other species

During sound production in canaries we measured peak expiratory flow rates of 1-7 ml/s and peak air sac pressures of 5-32 c m H 2 0 . Most avian vo- calizations examined have peak air sac pressures within the range of those recorded during canary song. Examples include starling (Sturnus vulgar&)

R.S. Hartley and R.A. Suthers: Respiratory dynamics in a singing bird 25

distress calls (18-25 c m H 2 0 ) , goose (Anser anser) honks (25 cm H 2 0 ) , grey swiftlet (Collocalia spo- diopygia) clicks (17 cm H/O), and oilbird (Steator- nis caripensis) clicks (10-30 c m H 2 0 ) (Gaunt et al. 1973; Brackenbury 1978a; Suthers and Hector 1982, 1985). Non-song vocalizations accompanied by peak air sac pressures similar to those recorded in canaries are listed by Gaunt (1987) for nine ad- ditional species. Crowing chickens are the excep- tion with very high air sac pressures of 50-60 cm H 2 0 (Gaunt et al. 1976; Brackenbury 1978a). Air- flow rate measurements have been made in fewer species, most of which have expiratory flow rates higher than those seen in canaries. Examples in- clude distress calls in starlings (10-15 ml/s) and evening grosbeaks (Hesperiphona vespertina) (17 ml/s), crowing in cocks (500 ml/s), honking in geese (650 ml/s), and click production in swiftlets (6-10ml/s) and oilbirds (17-250ml/s) (Gaunt et al. 1973; Berger and Hart 1968; Brackenbury 1978a; Suthers and Hector 1982, 1985).

Respiratory patterns in other species

Other studies have investigated the respiratory pat- terns that accompany non-song vocalizations in birds. Most trilled or pulsed sounds used the pulsa- tile expiration pattern. The ' churr' call of the star- ling, distress calls of the evening grosbeak, honking in the goose, and whinnies in the ring dove (Strep- topelia risoria) were all produced during pulsatile expiration (Gaunt et al. 1973; Berger and Hart 1968; Brackenbury 1978a; Gaunt etal. 1982). Clucking in chickens may be accompanied by mini- breaths in a few cases. In 20 out of 100 bursts of clucks, Gaunt et al. (1976) recorded a negative air sac pressure between consecutive sounds. They did not measure airflow, however, and so were un- able to determine the respiratory pattern. Bracken- bury (1978a) recorded tracheal airflow in clucking chickens and found, in some cases, what appeared to be inspiratory flow between successive notes. He likened this respiratory pattern to panting (both occur at 4-5 Hz) and did not call it 'mini- breaths'. Oilbirds consistently used the mini- breath respiratory pattern to produce long trains of sonar clicks at rates of 9-10 Hz (Suthers and Hector 1985).

Based on song recordings alone, Brackenbury (1978b) proposed that grasshopper warbler (Lo- custella naevia) song is accompanied by mini- breaths. These birds sing for up to a minute with- out interruption, their song consisting of rapidly repeated (26 Hz) note pairs. However, Schild (1986) examined several recordings of grasshopper

warbler song and found a few cases where the spec- trograms appeared as if two warblers were singing simultaneously, each with a slightly different note repetition rate. Because the 'inspiratory' and 'ex- piratory' phases (as proposed by Brackenbury) of the two song parts were not synchronous, Schild rejected the mini-breath hypothesis. It is unclear how the grasshopper warbler can maintain such a long duration song if it does not use mini- breaths.

Despite this result, we feel it is likely that song- birds with trilled or warbled songs use the mini- breath respiratory pattern. Males that sing for ex- tended periods are the most likely candidates. Al- though spectrograms are useful for studying bird song, physiological variables must also be mea- sured before one can determine how vocalizations are produced.

Acknowledgements. We thank Dr. F. Nottebohm for donating canaries, Dr. D. Hector for his technical advice, and the review- ers for their helpful comments on the manuscript. This research was supported by NSF grant BNS 85-19621 to R.A.S.

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