Brathing Pattern Co2 Elimination in Diving Weddell Seals

8/13/2019 Brathing Pattern Co2 Elimination in Diving Weddell Seals

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Respiratory Physiology & Neurobiology 162 (2008) 8592

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Respiratory Physiology & Neurobiology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / r e s p h y s i o l

Breathing pattern, CO2elimination and the absence of exhaled NO in freely

diving Weddell seals

K.J. Falke a,, T. Busch b, O. Hoffmann c, G.C. Liggins d, J. Liggins d, R. Mohnhaupt a, J.D. Roberts Jr. e,K. Stanek e, W.M. Zapol e

a Klinik fur Anaesthesiologie und Operative Intensivmedizin, Charite Campus Virchow Klinikum, Universitaetsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin, Germanyb Klinik und Poliklinik fuer Anaesthesiologie und Intensivtherapie, Universitaetsklinikum Leipzig, D-04103 Leipzig, Germanyc Klinik und Poliklinik fuer Neurologie, Charite Campus Mitte, Universitaetsmedizin Berlin, D-10098 Berlin, Germanyd Research Centre in Reproductive Medicine, Department of Obstetrics and Gynaecology, University of Auckland, Auckland 1142, New Zealande Department of Anesthesia and Critical Care, Harvard Medical School, Massachusetts General Hospital, 55 Fruit Street, Boston 02114-2696, MA, USA

a r t i c l e i n f o

Article history:

Accepted 15 April 2008

Keywords:

Marine mammals

Respiratory function

Lung volume

Lung collapse

Apnea

NO

a b s t r a c t

Weddellsealsundergo lung collapse duringdives below 50m depth. In order to explore thephysiological

mechanisms contributing to restoring lung volume and gas exchange after surfacing, we studied venti-

latory parameters in three Weddell seals between dives from an isolated ice hole on McMurdo Sound,

Antarctica.

Methods:Lung volumes and CO2 elimination were investigated using a pneumotachograph, infrared gas

analysis, and nitrogen washout. Thoracic circumference was determined with a strain gauge. Exhaled

nitric oxide was measured using chemiluminescence.

Results: Breathing of Weddellseals wascharacterized by an apneusticpatternwith end-inspiratory pauses

with functional residual capacity at the end of inspiration. Respiratory flow rate and tidal volume peaked

within the first 3 min after surfacing. Lung volume reductions before and increases after diving were

approximately 20% of the lung volume at rest. Thoracic circumference changed by less than 2% during

diving. The excess CO2 eliminated after dives correlated closely with the duration of the preceding dive.Nitric oxide was not present in the expired gas.

Conclusion:Our data suggest that most of the changes in lung volume during diving result from com-

pression and decompression of the gas remaining in the respiratory tract. Cranial shifts of the diaphragm

and translocation of blood into the thorax rather than a reduction of thoracic circumference appear to

compensate for lung collapse. The time to normalise gas exchange after surfacing wasmainly determined

by the accumulation of CO2 during the dive. These findings underline the remarkable adaptations of the

Weddell seal for restoring lung volume and gas exchange after diving.

2008 Elsevier B.V. All rights reserved.

1. Introduction

The extreme diving capabilities of Weddell seals (Leptonychotes

weddelli) are due to their high storage capacity for oxygen (Qvist et

al., 1986; Guyton et al., 1995; Kooyman and Ponganis, 1998) and its

remarkably economicalutilization mediated bythe diving response

(Scholander, 1940; Butler and Jones,1997). In addition the manage-

ment of pulmonary gas is paramount for efficient foraging in these

animals. They exhale before and immediately after the dive, char-

acteristics of their breathing pattern are obviously important for

the handling of the considerable amount of pulmonary nitrogen as

Correspondingauthor at:Am Neuen Garten 41, D 14469Potsdam, Germany. Tel.:

+49 331 249003; fax: +49 331 249005.

E-mail address: [email protected](K.J. Falke).

well as the regulation of buoyancy. So far only very few quantita-

tive studies on the pattern of respiration before and the restoration

of lung volume after free dives have been performed (Kooyman

et al., 1971, 1973; Parkos and Wahrenbrock, 1987). As originally

suggested byScholander (1940)and in line with Kooyman et al.

(1972)study in restrained seals, we have previously demonstrated

indirect evidence (by determining blood nitrogen tensions) that

the lungs of freely diving Weddell seals undergo alveolar collapse

during descentbetween 25 and50 m, a phenomenon stopping pul-

monary gas exchange and, thus protecting these animals from the

unwanted effects of excess nitrogen absorption (Falke et al., 1985).

However, directly measured data on the mechanical events associ-

atedwith lung compression, decompression and full recruitment at

surfacing aftereach divehave been extremely scanty.In a freely div-

ing trained dolphin, visible deformation of the chest wall began to

occur between 10 and60 m depth,and a photograph taken at 300m

1569-9048/$ see front matter 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.resp.2008.04.007
http://www.sciencedirect.com/science/journal/15699048mailto:[email protected]://localhost/var/www/apps/conversion/tmp/scratch_1/dx.doi.org/10.1016/j.resp.2008.04.007http://localhost/var/www/apps/conversion/tmp/scratch_1/dx.doi.org/10.1016/j.resp.2008.04.007mailto:[email protected]://www.sciencedirect.com/science/journal/15699048


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86 K.J. Falke et al. / Respiratory Physiology & Neurobiology 162 (2008) 8592

depth showed marked thoracic compression from just behind the

flippers and posteriorly (Ridgway et al., 1969).Following up on a

suggestion of the late Professor Hermann Rahn (personal commu-

nication), we attempted to correlate the changes in lung volume

during diving with measurements of thoracic circumference.

The aerobic diving limit (ADL) of Weddell seals is sufficient

(1520 min) for them to perform most underwater activities under

aerobic metabolic conditions (Kooyman et al., 1983; Qvist et al.,

1986; Castellini et al., 1992; Ponganis et al., 1993). In prolonged

dives, however, the arterial blood oxygen tension (PaO2 ) may fall

below 20 mmHg (Qvist et al., 1986) suggesting that low PaO2 is

not necessarily the primary respiratory drive or the most impor-

tant factor in the regulation of diving behavior (Stephenson, 2005).

Instead,a profoundimpact of CO2 on respiration anddiving (Pasche,

1976; Parkos and Wahrenbrock, 1987)has been reported but CO2elimination was not quantified.

In humans, breath-holding causes a pronounced increase of

gaseous nitric oxide concentrations in the upper respiratory tract

(Gustafsson et al.,1991; Persson et al.,1990). By analogy we hypoth-

esized that after the prolonged apnea of diving Weddell seals may

also accumulate NO in the airways, and because of its potent selec-

tive pulmonary vasodilatory effects (Frostell et al., 1991)it might

contribute to the re-establishment of pulmonary perfusion after

recruitment of the lungs at surfacing.

In the present study, we aimed to extend previous observations

(Kooyman et al., 1971, 1972, 1973; Kooyman, 1981b; Parkos and

Wahrenbrock, 1987; Falkeet al., 1985; Qvist et al., 1986) on the res-

piratory functions of freely diving Weddell seals firstly focusing on

the respiratory pattern using measurements of respiratory flows as

well aslung volumes andits shiftsbetweendives.We quantified the

changes in lung volume before, after and between dives expecting

that the seal would decrease its lung volume significantly before

and restore it to a large extent during the first few breaths after

surfacing. We also expected that lung collapse during descent, and

recruitment during ascent, would be associatedwith distinct corre-

sponding changes in thoracic circumference, a hypothesis based on

the above mentionedobservation madein a dolphin by Ridgeway in1969. Secondly we measured exhaled CO2 and hypothesized that

post dive CO2 elimination (VCO2 ) should correlate with the dura-

tion of the dive, a phenomenon which to our knowledge has not

been previously reportedin unrestrained Weddellseals. Thirdly, we

hypothesized that NO should be present in the expired gas of seals

in particular after the long breath-hold associated with long dives.

Thus, we aimed to determine the concentration of endogenous NO

in exhaled respiratory gas upon resurfacing.

Due to the constraints of our permit and due to the specific

Antarctic environmental conditions our studies were restricted to

three animals, consequently, our results are presented individually.

These data were extremely difficult to obtain, hence, they should

be considered a pilot study and hopefully will serve to stimulate

future studies.

2. Materials and methods

2.1. Animal handling and instrumentation

Allseal studies wereconducted underUS NMFSmarine mammal

permit #600, and approved by the Massachusetts General Hospital

Subcommittee on Research Animal Care. The general experimental

procedure has been well described (Kooyman et al., 1971, 1973;

Falke et al., 1985;Qvist et al., 1986; Parkos and Wahrenbrock, 1987).

Five sub-adultmale Weddellseals withweights estimatedfrom 280

to 340 kg were captured near the Dellbridge Islands, the Erebus

Glacier Tongue, and Arrival Heights on Ross Island (approximately

78

South, 168

East) and were moved to a field site equipped with

a portable research laboratory located above a onem diameter man

made ice hole where pulmonary function studies could be carried

out between free dives.

After the induction of anesthesia a previously calibrated (using

a ruler), 3 mm thick silicone covered mercury strain gauge (cus-

tom made by K. Stanek) was fitted around the seals chest caudal

to the fore flippers near the maximum circumference. The strain

gauge produced an electrical resistance proportional to the tho-

racic circumference. The resistance was converted to a voltage

using a Wheatstone bridge circuit. The signal was amplified, con-

verted to a digital format with 8 bit resolution and transferred to

the random-access memory of a time-depth recording computer

(Wildlife Computers, Woodinville, WA, USA) which was fixed to

a rubber patch glued to the seals dorsal fur. Measured values of

thoracic circumference werestoredevery second, andvalues of sea-

water depth every 5 s. After complete recovery from anesthesia the

seal was released into a temporarily opened second ice hole next to

the mobile laboratory. At the termination of the studies, the seals

were allowed to exit the water, and all monitoring equipment was

removed from the animals. The seal was then released near its site

of capture. The latex patches were left to fall off during molting.

2.2. Studies of respiratory physiology

For the measurement of ventilatory parameters, the ice hole

inside the laboratory was closed with a plywood sheet leaving

a central hole (20cm diameter) covered with an acrylic dome

to which a two way giant non-rebreathing valve (Series 7200,

Hans Rudolph, Inc., Kansas City, MO, USA) was attached closely

resembling the setup described by Parkos, Wahrenbrock and

Pasche(Parkos and Wahrenbrock,1987; Pasche, 1976). We inserted

an appropriately sized pneumotachograph (PT 18, Jaeger GmbH,

Wuerzburg, Germany) between the acrylic dome and the non-

rebreathing valve (seeFig. 1). The dome, the pneumotachograph

and the non-rebreathing valve increased the seals anatomical dead

space by approximately 2.5L. Forre-breathing maneuvers the non-

rebreathing valve was replaced by a large three-way stopcock and

a meteorological latex balloon of180 cm length and a capacity of

Fig. 1. Schematic representation of experimental setup using a pneumotachograph

to measure inspiratory and expiratory respiratory flow between free dives of Wed-

dell seals while resting in a man made ice hole (modified from Parkos et al.). For

re-breathing maneuvers the Rudolph valve was replaced with a large latex balloon

fitted with an appropriately sized three way stopcock.


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K.J. Falke et al. / Respiratory Physiology & Neurobiology 162 (2008) 8592 87

50 L, hanging from the ceiling of the hut. Hence, the gas volumes

in the bag and their concentrations were determined at room tem-

perature. Due to the specific heating conditionsof the experimental

hut there wasa large temperaturegradientbetweenthe bottom and

the ceiling of the hut, thus the room temperature could not be pre-

cisely determined. Consequently gas volumes were not corrected

to STPD or BTPS.1

A small suction line was attached between the acrylic dome

and the pneumotachograph to measure CO2 concentration during

breathing, using a standard infrared gas analyzer(Normocap, Datex

Instruments, Helsinki, Finland). Respiratory flow rate and CO2con-

centrationwere recorded onlineand stored in a personal computer,

utilizing software specifically designed for our studies. Tidal vol-

ume and ventilation wereobtainedby integration of the flowsignal.

The pneumotachograph was calibrated before and after each set

of measurements using a 2 L acrylic syringe simulating expected

flow ranges. The CO2 analyzer was calibrated using a gas mixture

containing 5% CO2in dry air. Mean expired CO2concentration was

either obtained by electronic integration of the CO2 signal or in

mixed expired gas collected in the latex balloon.

Lung volume was determined by washout of the nitrogen which

had remained in the lung during diving or during breathing at rest

into a closed system with a fixed volume of 100% O 2(the latex bal-

loon was filled to 20, 30 or 40 L). Nitrogen concentrations at the

end of re-breathing were calculated based on measurements of O2concentrations using an electrochemical monitor (Ohio 500, Ohio

Medical Corporation, USA) and CO2 concentrations (Normocap,

Datex Instruments, Helsinki, Finland) in appropriate gas samples

obtained from the latex balloon. To calculate the lung volume at

the beginning of oxygen re-breathing (VL start), either at surfacing

after diving or at rest, and at the end of re-breathing procedures

(VL end) equations according toKooyman et al. (1971)were used.2

Nitric oxide (NO) concentration in exhaled gas was measured

using chemiluminescence analysis, a process in which NO is con-

verted to NO2in the presence of an excess of ozone. This chemical

reaction is associated with the production of infrared radiation,

the intensity of which is a measure of the NO concentration. Theinstrument used (CLD 700 AL, ECO-Physics, Duernten, Switzerland)

possessed a lower detection limit of 0.5 parts per billion (ppb) and

a rise time of 2 s. The adjustment of the zero-level is accomplished

via interruption of a beam of light between the reaction cham-

ber and a photomultiplier; hence, NO free gas was not required

for calibration. In order to scale the display, a test gas with a NO

content of 90 ppm (AGA, Bottrop, Germany) was used. Consider-

ing the high linearity of the apparatus, this calibration is sufficient

for all concentrations below 100 parts per million (ppm). Analog

output of the instrument was converted to digital signals which

were stored continuously by our data acquisition system for later

analysis.

2.3. Data presentation and statistical methods

Because of the small number of animals we studied we will

present mostly individual data. However, in our investigation of

the differences in thoracic circumference at various depths we

1 In these measurements the effects of temperature variation need to be con-

sidered as a source of error. Pneumotachographic measurements were conducted

at or near BTPS, while gases collected in the rebreathing bag for determination of

lung volume were obtained at ambient temperature (near 0 C at the floor and fre-

quently near 30 C at the top of the laboratory). However, since the gas volumes at

the beginning and at the end were determined at the same environmental condi-

tions this should not have affected the calculation of the dilutional lung volume to

a relevant degree.2

See onlineSupplement I.

Table 1

Biometric data

Seal Total length

[cm]

Maximum

circumference [cm]

Nose to maximum

circumference [cm]

Estimated

weight [kg]

C 229 158 91 260

D 231 180 79 345

E 224 160 89 261

employed Wilcoxons signed rank test for matched pairs (two

tailed). A correlation analysis of CO2 excess production at sur-

facing after diving with the duration or depth of the dive was

performed using the Spearman rank correlation coefficient. All cal-

culations were carried out using SPSS statistical software (version

6.0). Results withp-values of less than 0.05 were considered as sta-

tistically significant. Data in the text and tables are expressed as

meanS.D.

3. Results

3.1. Biometric data

Wecaptured 5 Weddell seals (AE),but physiological data couldonly be obtained from 3 seals CE. Presented in Table 1are their

body weights (estimated according to Castellini and Kooyman,

1990).3 Because application of the strain gauge was a new tech-

nique we obtained reliable thoracic circumference measurements

only in 2 seals (D and E).

3.2. Pattern of ventilation

We collected data during a total of 32 phases of breathing

between dives. Respiratory frequency, mean and peak expiratory

and inspiratory flow rates, mean and maximal volumes as well as

the ventilation of seals CE are shown inTable 2.Data in the left

columnof numbers (Resting)wereobtainedwhilethe seal restedin

the ice hole (mean valuesS.D.). The columns to the right (headedAfter dive, 1 to 15 post) show individual values from the first to

the 15th minute after the termination of a single dive, one selected

for presentation from each of the three seals because of their long

duration. The column on the far right (Before dive) shows val-

ues from the last minute preceding the following dive. In these

three seals, we observed wide ranges of respiratory parameters

with minute ventilation near and above 100L per minute during

the recovery phase from the long dives. Some of the post dive val-

ues that were collected from seals C and E were obtained during

re-breathing maneuvers immediately after surfacing lasting 2 or

1 min, respectively. This was associated with a significant degree

of CO2retention. In seal D the values were obtained during normal

breathing without re-breathing.

Representative individual respiratory flow patterns, tidal vol-umes and exhaled CO2 concentrations are displayed in Fig. 2A

and B.Fig. 2A shows respiratory flow pattern, tidal volumes and

expired CO2 concentrations while resting in the ice hole with the

seal almost fully immersed in sea water and immediately prior to a

dive. Characteristically, the respiratory cycle begins with an expira-

tion which abruptly terminates at a very high flow rate and which

is immediately followed by an inspiration. A post-inspiratory pause

precedes the nextrespiratory cycle. Under theseconditions inhaled

and exhaled tidal volumes are identical, but the shape of the flow

curves differed and generally peak expiratory exceeded peak inspi-

3 For seal A the performance of the computer was unsatisfactory. Seal B escaped

from the site of investigation before useful data were obtained.
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Table 2

Individual respiratory parameters

Seal C Resting After dive #7 (duration 43 min; unknown depth) Before dive #4

81 1 post 2 post 3 post 4 post 5 post 9 post 15 post 1 pre

Breaths [per min] 10 2.0 13 12 17 17 16 16 9 11

Exp. flow, mean [L/s] 4.9 0.5 4.6 4.2 5.4 5.5 5.6 6.0 5.3 4.6

Exp. flow, peak [L/s] 7.0 0.7a 8.1 6.0 6.0 6.0 6.2 8.1 7.5 6.8

Insp. flow, mean [L/s] 4.8 0.7 4.1 4.1 6.4 6.8 6.8 5.7 4.9 4.7

Insp. flow, peak [L/s] 6.2 0.8a 6.1 6.5 7.2 7.2 7.2 6.7 5.5 5.4

Tidal volume, me an [L] 3 .9 0.4 4.0 4.8 5.1 5.6 5.6 5.4 4.1 3.9

Tidal volume max [L] 4.5 0.6 4.5 5.2 5.6 6.0 6.0 6.3 4.7 5.4

Ventilation [L/min] 38.9 7.7 52.5 57.3 87.4 95.6 90.3 86.3 37.1 43.0

End-tidal CO2[%] 4.8 0.4a Re-breathing 7.2 7.8 7.5 5.5 4.2 4.0

Exp. CO2mean [%] 2.5 0.1 4.1c 4.0c 3.9 4.0 3.8 3.1 2.6 2.1

VCO2 [cm3/min] 981 223 2156 2270 3443 3822 3437 2661 967 910

Seal D Resting After dive #2 (duration 33 min; depth 98 m) Before dive #1


Breaths [per min] 4 1 14 13 13 13 12 7 5 9

Exp. flow, mean [L/s] 4.5 0.4 6.3 6.0 6.2 6.1 6.1 5.2 4.3 5.3

Exp. flow, peak [L/s] 6.3 0.3a 8.4 6.2 6.2 6.2 6.4 6.0 5.8 6.3

Insp. flow, mean [L/s] 4.1 0.4 6.1 6.7 6.8 6.6 6.4 5.1 3.9 5.1

Insp. flow, peak [L/s] 4.9 0.5a 6.5 7.4 7.4 7.1 7.0 5.5 5.4 7.9

Tida l volume me an [L] 5.1 0.6 8.4 8.9 9.1 8.9 8.4 5.0 5.0 7.0

Tidal volume max [L] 5.5 0.6 9.2 9.1 9.3 9.1 8.7 7.1 5.4 8.4

Ventilation [L/min] 23.2 7.3 118.3 116.3 117.8 115.3 101.7 45.1 25.2 66.2

End-tidal CO2[%] 5.2 0.1a 8.2 8.4 7.9 6.8 5.9 5.0 4.8 4.2

Exp. CO2mean [%] 3.0 0.4 4.8 4.7 4.4 4.0 3.5 2.8 2.5 2.8

VCO2 [cm3/min] 695 256 5625 5397 5102 4588 3553 1256 640 1864

Seal E Resting After dive #5 (duration 29 min; depth 194 m) Before dive #3


Breaths [per min] 6 2 13 13 12 12 12 6 11

Exp. flow, mean [L/s] 4.0 0.6 5.1 5.5 5.4 5.3 5.1 4.3 4.8

Exp. flow, peak [L/s] 5.8 0.6b 7.2 7.2 7.0 6.7 6.2 6.0 6.1

In sp. flow, mean [L/ s] 4. 2 0.7 5.9 7.2 6.8 6.5 6.1 4.8 4.1

Insp. flow, peak [L/s] 4.6 0.4b 7.2 7.6 7.5 6.9 6.5 5.4 4.9

Tida l volume me an [L] 5. 6 0.7 7.6 8.5 8.3 8.1 7.4 5.9 6.3

Tidal volume max [L] 6.3 0.8 8.7 8.9 8.5 8.4 8.0 6.9 7.9

Ven tilation [L/ min] 33. 2 15.0 98.9 110.6 99.2 97.0 89.0 35.2 69.8

End-tidal CO2[%] 5.1 0.6b Re-breathing 9.5 8.9 7.5 6.6 5.2 4.2

Exp. CO2mean [%] 3.1 0.2 5.3c 5.1 4.6 4.2 3.7 3.0 2.5

VCO2 [cm3/min] 1000 432 5199 5617 4555 4026 3298 1046 1714

Restingvalues aregivenas meanS.D. pooled fromalldivesof eachsealinminutes(Seal C,81 min; Seal D,77min;SealE,67 min) except iflabeledassuperscript a= maximum

ofn = 6 readings, b = maximum ofn = 7 readings c= extrapolated because of re-breathing.

ratory flow (see alsoTable 2).For the last breath prior to diving, in

this example the seal takes only a small inspiration presumably to

reduce the gas volume of the lung (and therefore buoyancy) before

diving. After each dive (seeFig. 2B) the first respiratory maneuver

ofthe sealis a small expirationof the gas which has remainedin the

airways during the dive. This initial expiration usually has a large

peak flow velocity, demonstrating that it may be released at a high

pressure. In addition Fig. 2B shows that within the first few breaths

after the dive the inspiratory tidal volumes exceed the expiratorytidal volumes, indicating rapid refilling of the pulmonary gas vol-

ume after diving. This phenomenon of refilling thelungsaftera dive

and reducing lung volume before a dive is again demonstrated in

Fig. 3showing tidal volume continuously measured between two

sequential divesto depthsof 50 and150m. This figure also provides

a running difference between inspiratory and expiratory tidal vol-

ume, thereby quantifyinglung volume changes after andbefore the

dives. Of note, major changes in lung volume only occur during the

first few inhalations after surfacing and with the last exhalation

before diving (Figs. 2 and 3).

The mean reductions in lung gas volume (determined by pneu-

motachograph) caused by exhalation before diving were in seal C

1.6L (n = 1), in seal D 0.6 to 2.1 L (n = 3), and in seal E 3.2 to

5.5L (n =5).

3.3. Lung volume estimation by N2dilution

Values for diving and resting lung volume in seal C were 11.1

and 13.3 L, in seal D 11.8 and 13.1 L, and in seal E 16.3 and 18.6L,

respectively. The mean difference between resting volume and div-

ing volume was found tobe 1.90.6 L, a magnitude similar tothe

values obtainedusing the pneumotachograph.When determined at

rest, lung volumewas comparable at the startand endof the closed

circuit re-breathing procedure (15.0L vs. 17.4 L). However, whendetermined after surfacing from diving, lung volume increased

markedly during the re-breathing procedure (VL start: 13.12.8L;

VL end: 24.412.4 L).

All measurements that were carried out simultaneously in seal

E are exemplified graphically in Fig. 4. It shows the CO2elimination

rate, measurements of lung volume and changes in lung volume

(equal to tidalvolume),thoracic circumference,and depth oversev-

eral sequential free dives. After dive #2 a re-breathing maneuver

was undertaken in order to determine the lungs gas volume by

N2dilution. In the example shown this led to the previously men-

tioned marked increase of lung volume which was confirmed by

the dilutional volume determination. Lung volume determined by

N2dilution at the beginning of re-breathing was 16 L andat the end

37 L, probably near the seals total lung capacity.


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Fig.2. (A)Respiratory gasflow,lung volume, tidalventilationand exhaled CO2con-

centration between and during the last respiratory cycles (seal E) before diving for

30min to a maximum depth of 194 m. Lung volume reduction prior to diving of

4 L (a, below interrupted line) was restricted to the last breath. Positive flow val-

ues correspond to the expiratory phase and negative to the inspiratory phase of

the respiratory cycle. Due to the measuring technique, the CO2

signal is delayed by

approximately 1.5s as compared with the gas flow recordings. (B) Respiratory gas

flow, lung volume (VL), tidal volume and exhaled CO2concentration of seal E during

the first breaths taken after surfacing from a dive of 12 min duration. The seal first

exhales a small gas volume (first small dip ofVL) a part of which had remained in

the airways and then re-inflates the lungs with the first two inhaled breaths. How-

ever the net-increase in lung volume by inhalation remains surprisingly small (a)

and corresponds to the decrease ofVL measured in the last breath before diving

(seeFig. 3).Hence, most of the restoration of lung volume must be due to decom-

pression of the gas (b) which had remained in the airways during the dive. It was a

generalobservation thatthe exhaled CO2concentration of thefirst fewbreaths after

returning to the surface was quite low and similar to resting levels.

3.4. Thoracic circumference changes

The thoracic circumference changes of seal E during diving as

measured by the strain gauge are shown in Table 3.We observedsteep changes of circumference with decreases at the beginning

of the dive and increases at the return to the surface (Table 3and

Fig. 4). Comparing the measurements immediately after the begin-

ning of the dive at 0 m (seeTable 3)and at maximum depth, the

mean difference in circumference was 2.580.55cm. The main

characteristics of circumferential data recordings are revealed by

inspection of an example in seal E (Fig. 4) which contains addi-

tional results of accompanying respiratory measurements covering

a period of several dives and rest phases. At approximately 50 m

depth (dive #2) when the lung is collapsed, there was a minimal

reduction of 1 cm in the thoracic circumference. Even at 150 m

depth (dive #2) the decrease was only in the range of 23 cm

(Table 3). The unexpected substantial increase in lung volume from

16 to 27L (reproduced by dilutional lung volume determination)

Fig. 3. As an example, the shifts in lung volume are shown between 2 dives. The

lung volume and the tidal volume are shown in the upper panel. The absolute lung

volume in the upper panel was taken from the N 2 dilution whereas the changesin volume and the tidal volume were obtained from the pneumotachograph. In the

lowerpanel, VinspVexp is shown demonstrating theincreasein lungvolumeimme-

diately afterthe dive (left), and thereductionof thelungvolumeoccurring with the

last expiration before the next dive (right). It demonstrates that only 2025% of the

alterations in lungvolumeoccur due to ventilation. A muchlargerproportion of the

diving lung volume is compressed.

Fig. 4. Computer tracingsdemonstrating the results of all measurementsthat were

carried outsimultaneously (seal E), from thetop down:breath bybreath CO2elimi-

nation(VCO2 ),lungvolume(VL), itschanges andtidal breaths,thoracic circumference

(Circ), anddepth during andat rest of severalfree dives. After dive #2 a re-breathing

maneuver was undertaken in order to determine the N2 dilutional lung volume. In

contrast to the volume recordings after dives #1 and 3 when no re-breathing took

place this was associated with a doublingof thelungvolumepresumably dueto the

inability of the seal to remove the CO 2. This is a true lung volume change (and no

artifact) becauseit wasconfirmed by theindependentdetermination ofVLby the N2dilution technique, calculating the volume at the beginning VL start= 16 L and at the

endVL end= 37L of the re-breathing maneuver, a level probably near the total lung

capacity. It is interestingbecause it supportsour notion thatchanges in lungvolume

(here in the opposite direction as compared to descent in the water) are associated

with very small almost not recognizable thoracic circumference changes (see

panel 3 from the top).


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Table 3

Thoracic circumference of seal E at various depths

Dive number Circumference Maximum depth of dive [m]

Before dive [cm] At 0 m [cm] At 10 m [cm] At 20 m [cm] At maximum depth [cm]

2 175.2 171.8 171.3 170.4 169.6 147

3 173.6 172.0 171.1 170.4 169.6 157

5 174.3 171.7 170.9 170.4 168.3 196

9 172.9 171.1 170.4 169.6 168.8 152

Mean 174.0 171.7 170.9 170.2 169.1 163.0

S.D. 1.0 0.4 0.4 0.4 0.6 22.4

which occurred during a re-breathing maneuver following dive

#2 also resulted in only a 1 cm increase of thoracic circumference

(Fig. 4, immediately following dive #2).

Our findings were further substantiated by comparing the cir-

cumference values measured when the curve stabilized shortly

after the beginning of the dives (with the seal at 20 m of depth)

with those measured at the deepest point of the dive. Including

data from 11 dives (3 from seal D and 8 from seal E) with a mean

depth of 17655 m (all greater than 82 m) we could demonstrate

only a small albeit significant decrease in thoracic circumference

(surface, 166.5

5.3 cm vs. depth, 165.3

5.3cm,p = 0.003).

3.5. CO2exhalation studies

The end-tidal as well as the mean expired CO2 concentrations

and the mean rate of respiratory CO2elimination (VCO2 ) are shown

in Table 2. Breathing at rest, the end-tidal (=maximal) expira-

tory CO2 concentrations ranged between 4.80.35 and 5.10.6%

while the basal CO2elimination rate varied between 695256and

1000432mL min1. After surfacing commonly three exhalations

were required to wash outthe artificialdead space andto reach CO2concentrationsreflecting alveolargas. The end-tidalCO2 concentra-

tions as well as the breath-by-breath CO2elimination increased to

maxima within the first 23 min after surfacing, up to 89% and

5.6Lmin1, respectively (Table 2) and approached normal rest-

ing values within 15 min (Figs. 4 and 5). After a total of 12 diveswe observed a significant and marked linear correlation (R = 0.73,

p = 0.007) between the CO2eliminated in excess of the basal elimi-

nationrate during rest andthe duration of thepreceding diveswith

values obtained up to >40 min of dive duration (Fig. 6).

3.6. Nitric oxide in exhaled gas

In the expiratory gas of all 5 seals, we could not detect any

exhaled NO, indicating that NO was either completely absent from

Fig. 5. Breath by breath CO2 elimination in a single seal after a dive to 48 m depth

for 12min duration. TheVCO2 of the first three breath is underestimated due to

the artificial dead space of the breathing dome. The dotted line indicates the CO 2elimination during rest. It was a common characteristic of our measurements that

the maximum amount of CO2 was exhaled within the first 23 min after surfacing.

Fig. 6. CO2 elimination in excess of the resting basal rate during diving (retained

CO2) as determined after surfacingand plotted againstthe durationof thepreceding

dive. A significant and close correlation is demonstrated (R = 0. 986,p


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pressure valves closing after inspiration (Scholander, 1940, p.

23).

An interesting question is the extent to which expiration is per-

formed as an active manoeuvre. The weight of the chest wall and

blubber as well as the pressure exerted by the surrounding sea

water would imply that expiration is mainly passive. The observed

expiratory flow pattern, however, differs from humans whose pas-

sive expiration is characterized by a decelerating flow. In contrast

we observed forceful increases and decreases in airway gas flow

with a plateau phase between them (Fig. 2), suggesting active mus-

cular forces are operating during expiration. This is in line with the

observation ofKooyman and Cornell (1981)of an extremely high

expiratory peak flow rate of 162 L s1 in a dolphin trained to make

a maximum expiratory effort.

It is well known (see Figs.2Aand3)that Weddell seals usually

exhale before diving (Kooyman et al., 1971), presumably in order to

decrease buoyancy and, thus facilitate their descent in the water.

It was an important goal of our studies to quantify the changes in

lung volume immediately before and after dives. In contrast to our

initial hypothesis, the degree of these lung volume changes was

relatively small. In the example shown inFig. 1Athis exhalation

before diving represents a lung volume reduction of approximately

only 20%. After thedivewhen theseal surfaces thefirstexpiration is

of a similar magnitude (see Figs.2Band3).This represents a com-

ponent of the gas, which has remained compressed in the upper

airway during the dive after lung collapse and is usually released

with considerable positive pressure. Presumably this gas, which is

considered a portion of the diving lung volume, re-expands with

the decreasing water pressure as the seal approaches the surface

and, hence, due to its evolving positive pressure contributes to the

recruitment of the previously collapsed lung. We also note that

complete restoration of lung volume to the pre-dive level occurs

quickly withinthe first 24 breaths after surfacing, most of it taking

place in the first breath (see Figs.1Band2).In the example shown

inFig. 1B, 16 L of gas are compressed to 2.7L (b) at 50m depth

which is forced into the upper airways indicating that lung collapse

occurs approximately at this depth or slightly beyond, an interpre-tation corresponding well with earlier observations (Kooyman et

al., 1971; Falke et al., 1985).

4.2. Circumference measurements

Lung collapse in diving Weddell seals has been explained by a

compression of the chest wall (Kooyman, 1981a,b; Kooyman and

Ponganis 1998), similar to observations in freely diving dolphins

(Ridgway et al., 1969;Ridgwayand Howard,1979). According to our

previous studies, complete lung collapse should occur at 2550 m

below the surface (Falke et al., 1985). Surprisingly at this range

of depths we consistently observed only a very small decreasein thoracic circumference of about 23 cm (i.e., less than 2%). In

principle, irregular deformation of the thorax could occur without

marked changes at the level of our circumference measurements,

but it is more reasonable to assume that the lack of major changes

of thoracic circumference is due to additional mechanisms going

along with the marked decrease in intrathoracic volume such as

an upward movement of the diaphragm as well as the displace-

ment of blood from extrathoracic to the intrathoracic vasculature

(Kooyman, 1981a,b).

Acalculationusingaconeasamodelofthesealsthorax(withthe

tip being the nose and the base the equivalent of the diaphragm)

revealed that the diaphragm would be shifted upwards within a

plausible range of 512 cm if the determined diving lung volumes

and the observed small changes in thoracic circumference were

taken into account.4 There is evidence from human (Craig, 1968;

Schaefer et al., 1968) and animal diving observations (Cozzi et

al., 2005; Ninomiya et al., 2005)that translocation of blood from

the peripheral to intrathoracic/-pulmonary vessels may also play a

space filling function when the seals lung collapses.

It should, however, be stressed that thoracic circumference is

probablynot onlyaffectedby passive compression of the chestwall,

but also by muscular activity. Since the strain gauge was placednear the flippers, we believe that the sharp changes in measured

circumference immediately at the beginning and at the end of a

dive (seeFig. 4)could well be caused by activation of the flippers.

4.3. CO2concentration and elimination

Due to the configuration of our measurement device (dome

and pneumotachograph) the anatomical dead space was artificially

increased by approximately 2.5 L. AssumingPCO2 being theprimary

drive for ventilation (Parkos and Wahrenbrock, 1987) this addi-

tional dead space should slightly increase minute ventilation and

decreasemeanexpired CO2concentration accordingly. Due to this

effect the end-tidal CO2 underestimated alveolar PCO2 and theVCO2

of the first three breath after surfacing (Table 2and Figs.2Band5).Taking into account the large lung and tidal volumes of the animals

in particular aftersurfacing the additional deadspace was,however,

readily washed out within three breaths as demonstrated by the

original CO2recordings (partially shown inFig. 2B). In our opinion

it becomes insignificant during subsequent exhalations and during

steady state conditions (resting) in respect to the determination of

the VCO2 which was the main focus of our investigation. Our end-

tidal CO2 concentrations were in a range close to these previous

observations of end-tidal CO2and blood gas tension levels (Parkos

and Wahrenbrock, 1987; Qvist et al., 1986).

Because we had to rely on the estimated body weights of just

three animals we hesitatedto presentVCO2perkg body weight.Nev-

ertheless we found an excellent and statistically highly significant

correlation between dive duration and post-dive CO2

elimination

above the basal rate. Breath by breath analysis of CO 2 elimination

showed that the elimination of retained CO2 reached a maximum

after 14min with a 38-fold increase inVCO2above resting values.

It is notable that the time course of the CO 2 elimination pro-

file resembles that of the end-tidal O2 concentration observed by

Ponganis et al. (1993).

4.4. Nitric oxide

Gaseous nitric oxide (NO) a potent selective pulmonary

vasodilator produced in the epithelium of the lower and upper res-

piratory tract (Busch et al., 2000),and primarily in the paranasal

sinuses (Lundberg et al., 1995; Deja et al., 2003) is auto-inhaled

in humans (Gerlach et al., 1994; Lundberg et al., 1996)and many

terrestrial animals (Gustafsson et al., 1991; Jenkins and Langlois,

1995;Lewandowski et al., 1996;Schedin et al.,1997). After a human

breath-hold the exhaled concentration may be as high as 30 ppb

(Kimberly et al., 1996).Accordingly, we hypothesized that gaseous

NO should accumulate in the upper respiratory tract of diving

Weddell seals and by its auto-inhalation might contribute to the

restoration of pulmonary blood flow at the end of the dive. How-

ever, we could not detect any NO in the expiratory gas of Weddell

seals.Due to ourlower detectionlimitof 0.5ppb we cannotrule out

even lower concentrations in the parts per trillion range. After ter-

mination of our studies, based on measurements in baboons it was

4

The detailed description of the model calculation, see onlineSupplement II.
http://-/?-http://-/?-http://-/?-http://-/?-


8/8


hypothesized (Lewandowski et al., 1998) that terrestrial animals

without paranasal sinuses do not have NO concentrations above a

few ppb in their respiratory gas. Hence, we believe that the main

explanation for the absence of NO in the exhaled gas of Weddell

seals is their lack of open and aerated paranasal sinuses ( Negus,

1958; King, 1983).Other factors may include scavenging of NO by

blood hemoglobin which increases to levels around 25 g% during

diving in Weddell seals (Qvist et al., 1986).

Acknowledgements

Thesestudieswere fundedby NationalScience Foundation grant

OPP 91-18192 and also supported by the Deutsche Forschungsge-

meinschaft, grant DFG Fa 139/4. We thank Bernhard Huettel, the

German representative of ECOPhysics (Duernten,Switzerland)who

provided theequipment for theNO measurementsand Axel Mohn-

haupt who provided the computer software. Special thanks goes to

Professor PeterScheid forhis very valuablesupport in finalizingthe

manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,

in the online version, atdoi:10.1016/j.resp.2008.04.007.

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Brathing Pattern Co2 Elimination in Diving Weddell Seals