The Regulation ofBreathing in the Chick Embryo
By
Tara Marisa Menna
Department ofPhysiology, McGill University, Montreal, Quebec December, 2001
A thesis submitted to the Fàculty of Graduate Studies and Research in partial fulfillment of the requirements ofthe degree of Masters of Science.
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Canada Abstract - From the onset ofintemal pipping (i.e. embryonal breathing ofair cell gas) until hatching, the chorioallantoic membrane (CAM) and the lungs work simultaneously to serve the metabolic needs ofthe embryo.
1. The carbon dioxide and oxygen exchange rates (V02and VC02) through the lungs and
the CAM were separately, but simultaneously, measured during the last two days of
incubation (day 20-21), while ventilation (VE) was calculated from the measurements
ofpressure oscillations in the air cell. When the embryo's total metabolic rate was
increased, VE was linearly proportional to lung V02 and VC02 and not to the embryo's
total metabolic rate.
2. Tracheal pressure and changes in lung volume were quantified through mechanical
ventilation ofthe embryo. The curled up posture ofthe embryo, the eggshell and its
membranes did not represent a significant mechanical constraint to VE.
3. VE, lung VÛ2 and VC02 were measured while the CAM compartment was exposed to
either 10% O2, 100% O2 or 5% CO2. Total V02was also measured u.nder these
conditions. There is a clear VE-sensitivity to CO2and a rather weak VE-sensitivity to
changes in arterial oxygenation present at this stage ofdevelopment. ii
Résumé - Dès les débuts des 'pipping' internes jusqu'à l'éclosion de la coquille, la membrane chorioallantoique (MCA) et les poumons travaillent simultanément afin de servir les besoins métaboliques de l'embryon.
1. Les taux d'échanges du gaz carbonique et de l'oxygène (Vo2et VC02) à travers les
poumons et la MCA furent mesurés séparément, mais simultanément durant les deux
derniers jours d'incubation (jour 20-21), pendant que la ventilation (VE) fut calculée
basé sur les mesures des oscillations de pression dans la cellule d'air. Lorsque le total
du taux métaboliques fut augmenté, VE fut proportionnelle liniairement à V02 et au
VC02 des poumons et non au total du taux métaboliques de l'embryon.
2. La pression tracheal et les changements de volume dans les poumons furent évalués
quantativement à travers la ventilation mécanique de l'embryon. La posture
recroquevillée de l'embryon, la coquille and ses membranes n'ont pas apporté de
contraintes mécaniques importantes à la VE.
o 0 0 3. VE, V02et VC02 des poumons furent mesurés pendant que le compartiment de la
MCA fut exposé à 10% O2, 100% O2ou 5% C02. Le total de V02fut également
mesuré avec ces conditions. TI est évident que la VE est sensible au C02. mais
parcontre peu sensible aux changements d'oxygène dans le sang qui existe à ce stade. III
Contributions of Authors
Dr. Jacopo P. Mortola contrived the experimental protocol carried out in Chapters
2, 3 and 4 and contributed to the design of the various setups used for the measurements of chick embryo pulmonary ventilation, gas exchange and pulmonary mechanical properties. Lina Naso conducted the experiments required to construct Figure C.I (see
Appendix C). iv
Acknowledgements
AlI ofthe experiments in Chapter 2 and 4 were financiaHy supported by the
Medical Research Council ofCanada. The experiments conducted in Chapter 3 were assisted by funds from the Canadian Institute ofHealth Research. The studies were conducted in agreement with the current Canadian regulations on Animal Ethics.
1 would like to take this opportunity to thank the foHowing people for making my journey as a graduate student such an enriching experience. First and foremost, my supervisor, Dr. Jacopo P. Mortola. Thank you for aH ofyour help, support, guidance, wisdom and patience. You are an incredible teacher and a true inspiration. 1 have learnt so much from you and hold you in the highest respect. To the lab technician, Lina Naso, my dear friend, thank you firstly for conducting the western blot experiments on my behalfand secondly, for your constant encouragement. You have a superb ability to find the solution to any problem and 1 truly admire that. You undeniably run the labo Erin
Seifert and Dr. Gilherme Santanna, it was always refreshing to know that 1 was never alone when times got hard. Thank you for being there.
Michèle Tredger, thank you for the time you spent translating my abstract. Eric
Jr. Pauyo, thank you for your solid advice and for sharing your experiences with me.
Lastly and most importantly, 1 would like to dedicate my efforts to my family.
Mom, Dad and Eric, 1 hope 1 make you proud. Thank you for your love, generosity and understanding. v
Abbreviations
See also the Glossaryfollowing the text
CAM chorioallantoic membrane
C02 carbon dioxide
Crs respiratory system compliance f breathing frequency
02 oxygen
P pressure
Pa co2 arterial partial pressure ofcarbon dioxide
P02 partial pressure ofoxygen
QlO an index oftemperature sensitivity. See a/sa Glossary
Rrs respiratory system resistance
TI inspiratory time
V volume
0 VC02 carbon dioxide production
0 VE pulmonary ventilation
0 V02 oxygen consumption
VT tidal volume us time constant ofthe respiratory system
Zrs respiratory system impedance vi
Table of Contents
1. Abstract i ll. Résumé , .ii ill. Acknowledgements iii IV. Contributions ofAuthors .iv V. Abbreviations , , , , v
1. Introduction 1
1. 1. Dual Gas Exchangers 2
1.2. The Eggshell ,, , 3
1.3. Gas Exchange across the Eggshell 3
1.4. The Transition to Lungs , 6
1.5. The Pulmonary Respiratory System ,, 7
1. 6.Project Objectives 8
2. Metabolic Control ofPulmonary Ventilation in the Developing Chick Embryo.11
2.1. Abstract 12
2.2. Introduction 13
2.3. Materials andMethods 14
2.3.1. Preparation 15
2.3.2. Temperature Equilibration 16
2.3.3. Gaseous Metabolism 17
2.3.4. Pulmonary Ventilation...... 18
2.3.5. Protocol andData Analysis 19
2.4. Results 20
2.5. Discussion 22 vii
2.5.1. Temperature andEmbryo 's Metabolism 23
2.5.2. Breathing Activity 24
2.5.3. Breathing and Changes in Egg Temperature 25
3. Respiratory Mechanics in Chick Embryos Before and After Exteriorization
From the Eggshell .29
3.l.Abstract , 30
3.2. Introduction 31
3.3. Materials andMethods 32
3.4. Results 34
3.4.1. Internai Pippers 34
3.4.2. External Pippers andHatchlings 35
3.5. Discussion 35
3.5.1. Comparison with Mammals andAdult Birds 35
3.5.2. Internai Pippers 37
3.5.3. External Pippers 38
3.5.4. The SealedEgg 38
4. Ventilatory Chemosensitivity in the Chick Embryo .41
4.1. Abstract ,, ,, 42
4.2. Introduction .43
4.3. Materials andMethods .44
4.3.1. Total Gaseous Metabolism .45 viii
4.3.2. VE andV02Iung .46
4.3.3. Protocol andData Analysis 48
4.4. Results 49
4.4.1. Total Metabolism 49
4.4.2. Pulmonary Responses to Hypercapnia .49
4.4.3. Pulmonary Responses to Changes in Oxygen 50
4.5. Discussion 50
4.5.1. Effects ofchanges in oxygenation onMetabolism 50
4.5.2. Ventilatory responses to changes in oxygen 52
4.5.3. Response to hypercapnia 53
5. Summary and Conclusions ,, , 56
5.1. An Overview ofthe Mode!. , , 57
5.2. The Goals ofthe Study 58
5.3. The Metabolic Aspect " , 58
5.4. The Mechanical Aspect 60
5.5. Chemosensitivity 61
Appendix A: Effects ofTemperature on Oxygen Consumption throughout Incubation..64
Appendix B: The Barometric Method. , 67
Appendix C: Identification ofUCP , 71
Glossary 72
Reference List 76 Chapter 1
Introduction 2 1. Introduction
1.1. Dual Gas Exchangers
Alterations in pulmonary ventilation tightly follow the alterations in metabolic rate. This coupling has been demonstrated multiple times over the years in severa! conditions, for example, during hypermetabolic activity like exercise, thermogenesis or from pharmacological interventions and as well during hypometabolic activity, such as exposure to hypoxia (Mortola and Gautier, 1995). The mechanisms responsible for the close link between metabolism and pulmonary ventilation remain unclear.
In mammalian physiology, it is difficult to explore the relationship between these two parameters because ofthe simplicity ofthe respiratory system design, that is, having a sole gas exchange unit, the lungs. The lungs provide the only possible pathway for oxygen to be taken in and for carbon dioxide to be expelled out into the environment. The question arises therefore, what ifa secondary gas exchanger is brought into play as an alternate route for gas exchange? How is ventilation affected by changes in metabolism in an animal that does not fully depend on its lungs for gas exchange?
There are essentially no mammals that normally provide this setup ofa dual gas exchange system, with the exception ofthe newborn marsupial, where at birth, skin breathing seems to be the predominant form ofgas exchange (Frappell and Mortola,
2000). There are sorne lower vertebrates, such as amphibians and salamanders that also possess the ability to breathe through the skin (Gatz, 1982). Alternatively, nearing the end ofincubation, the avian egg, for instance the domestic fowl egg, provides a situation 3 ofdual gas exchange, whereby the chorioallantoic membrane and the lungs function side by side to meet the metabolic needs ofthe embryo.
1. 2. The Eggshell
The egg is comprised ofaIl the nutrients necessary for embryonic growth with the exception ofoxygen. The eggshell is composed ofcalcium carbonate in the form of calcite columns. During the formation ofthe shell, the geometry ofthe crystal columns allot for sorne small spaces to remain open, thus forming pores that cross the shell radially (Wangensteen and Weibel, 1982). The porous nature ofthe shell, therefore, allows gases to travel through it following the simple laws ofdiffusion (Tullett and
Deeming, 1982). The permeability ofthe eggshell, which can be measured most simply as shell conductance, is dependant upon the size and number ofthe pores. On average, the domestic fowl egg is comprised of 10,000 pores, each approximately 0.3mm in length, with a mean cross-sectional area ofabout Il0llm2 (Burton and Tullett, 1985).
1.3. Gas Exchange Across the Eggshell
In the domestic fowl, incubation lasts approximately 21 days. The principal respiratory organ for the majority ofthe incubation is the highly vascular chorioallantoic membrane (CAM). It begins to develop at day 4 ofincubation when the chorionic epithelium fuses to the vascular allantoic membrane. The membrane continues to extend its area until, by day 12, its capillary network covers the entire inner surface ofthe egg, just beneath the outer and inner shell membranes. These two shell membranes are essentially one fibrous compound lining the inner egg surface exclusive ofthe blunt end 4 ofthe egg. At this junetion, the two membranes separate and form an air poeket, or what is termed the air cell. The air cell initially develops shortly after the egg has been laid due to the warm contents ofthe egg contracting when exposed to the cooler ambient environment. Consequently, due to the porosity ofthe shell, there is a continualloss of water from the egg as it escapes by diffusion as vapor. Therefore, the air cell continues to grow in size throughout the incubation time.
An ongoing debate reflects the adaptation ofshell porosity to balance the dual role ofmeeting the metabolic requirements ofthe embryo with that ofpreserving water losses. For example, larger eggs can acquire more oxygen by having a more porous shell
(in both number and size); however, this leads to an increase in water loss from the egg and too much water loss during incubation leads to dehydration ofthe embryo. It has been suggested that shell conductance is determined primarily to regulate water loss
(Tullett and Deeming, 1982). Porosity is increased in humid environments, whereas the opposite occurs in dry conditions. At increasing altitudes, barometric pressure decreases, thus increasing the rate ofdiffusion ofgases because ofthe inverse proportionality between barometric pressure and gas diffusion. Up to a given altitude, egg shell porosity is reduced to limit water loss; at yet higher altitudes, egg shell conductance conversely increases to compensate for the low oxygen pressure (Black and Snyder, 1980; Monge et al., 1997).
During a natural incubation, eggs lose, on average, 15% oftheir freshly laid mass; this value is essentially constant despite species differences in incubation time or egg size
(Burton and Tullett, 1985). An intriguing observation is that avian embryos have the ability to adapt to a vast range ofeggshell porosities and incubation conditions. The 5 resulting variability in water loss among eggs can be accommodated for the most part because there are water reserves within the egg and it is only until these reserves are used up that the effects ofwater loss begin to affect the embryo directiy. Embryos can adapt to low porosity shells by reducing their metabolism and increasing their incubation time
(Wangensteen and Rahn, 1970/71).
Respiratory gas exchange between the environment and the CAM takes place by diffusion across severallayers ofresistance. These layers consist ofthe sheIl, the outer and inner shell membranes and the chorioallantoic endothelium. At the beginning of incubation, the inner shell membrane is the major site ofresistance. However, due to the progressive removal ofwater from in and around the membrane fibers, the resistance dramatically drops by the end ofincubation. At the end stage ofdevelopment, the largest resistance is accounted for by the rate ofoxygen binding to hemoglobin at the level ofthe chorioallantoic capillaries (Wangensteen and Weibel, 1982).
The embryo and the CAM interact via the allantoic artery and allantoic vein. That is, oxygen-poor and carbon dioxide-rich blood leaves the body through the allantoic artery and enters the capillary network ofthe CAM. Here, the blood takes up oxygen that has diffused into the shell in exchange for the endogenously produced carbon dioxide.
The newly oxygenated blood retums to the embryo by the allantoic vein (Tazawa et al.,
1983).
As the oxygen demands ofthe embryo increase, the chorioallantoic blood undergoes progressive changes to accommodate the needs accordingly. For example, the total amount ofhemoglobin increases in the circulation due to an increase in the number red blood cells. This leads to an equivalent increase in oxygen capacity. As weIl, the 6 blood's oxygen affinity increases due to the depleting stores ofintra-erythrocytic ATP and as a result, chorioallantoic blood continues to be approximately 90% saturated with oxygen despite the falling partial pressure ofoxygen in the air cell (Burton and Tullett,
1985). This alteration helps in providing sufficient O2 transport to the embryo.
1.4. The Transition to Lungs
When an egg is laid, the porosity ofthe shell is fixed. Therefore, as the metabolic needs ofthe embryo increase beyond the CAM's diffusive capabilities, the conditions in the egg become increasingly hypoxic and hypercapnie. The diffusive process alone could not provide the energy necessary for hatching; thus, at the end ofincubation, pulmonary ventilation must be initiated (Visschedijk, 1968a; Pettit and Whittow, 1982). Gas exchange can now take place in the CAM as well as in the lungs, thus alleviating the limitation offered by the shell.
Toward the end ofincubation, at around day 20 in the domestic fowI, the beak of the embryo pierces through the inner shell membrane and begins to breathe air cell gas.
This effort is termed internaI pipping. From this moment on, pulmonary respiration continues to increase and predominates as the primary gas exchange organ, while gas exchange through the chorioallantois gradually declines as blood is withdrawn from the membrane (Ar et al., 1980). Therefore, a transition takes place whereby diffusive gas transport via the CAM is gradually replaced by convective gas transport via the Iungs.
This process is even more accelerated after external pipping, where the beak cracks the eggshell and is now exposed to atmospheric air (Vince and Tolhurst, 1975). Hence, 7 during this period oftransition, the embryo utilizes two respiratory gas exchange organs simultaneously to meet the metabolic requirements needed for a successful hatch.
1. 5. The Pulmonary Respiratory System
The bird respiratory system can be illustrated most simply as comprised ofthe lungs, the airsacs and the chest wall. On day 4 ofincubation in the fowl egg, the lungs and the airsacs begin to develop. Each lung consists ofa primary bronchus, which further gives rise to a series of secondary bronchi, which extend again into the tertiary bronchi, also named the parabronchi. The parabronchi elongate and form a tubular network. Eventually, these give rise to air capillaries, which in turn become intimately associated with blood capillaries to form the gas exchange area. Bronchial connections to the airsacs sprout trom the primary bronchus. The airsacs slowly develop and eventually expand on both sides ofthe lungs (Duncker, 1978). The airsacs are thin-walled structures lacking significant vasculature; however, they are responsible for driving air through the lungs. Their function is critical to the respiratory system sinee, differently trom mammals, the lungs ofbirds are relatively stiffand non-expansible. For example, Jones et al. (1985) found that the lungs ofducks expand by less than 1% during a regular breath.
Two or three days prior to hatching, the embryo ingests the amniotic fluid and the remainder ofthe albumen into its gut. Although the space between the embryo and the inner shell membrane is now aerated, its minute volume is not advantageous with regard to functional pulmonary respiration. Nevertheless, several studies have shown that respiratory movements do indeed begin before the onset ofinternaI pipping (Vince and 8 Tolhurst, 1975), a process analogous to the breathing movements ofthe mamma1ian fetus. These movements are said to possibly facilitate the later aeration ofthe system by improving the removal and absorption ofliquid from the fluid filled structures. Aeration ofthe respiratory system is graduai and often takes several hours (Visschedijk, 1968a).
The mechanisms responsible for the initiation ofthe first breath continue to remain debatable. The high partial pressure ofcarbon dioxide and, as weIl, although not as effective, the low partial pressure ofoxygen found in the air cell near the end of incubation are thought to influence the timing ofpipping (Visschedijk, 1968b). In addition, the boost in circulating thyroid hormones at this stage is also believed to play a potential role (Burton and Tullett, 1985).
As a result, this naturally occurring, unique situation ofsimultaneous gas exchange between two respiratory organs offers a novel opportunity to explore the relationship between metabolism and pulmonary ventilation. In addition, a more specifie look into the control ofbreathing in the avian embryo is also ofinterest given that few data are available regarding ventilatory regulation at this stage ofdevelopment.
1. 6. Project Objectives
Given this model, several intriguing questions arise. For instance, when metabolism changes in an animal with two functioning respiratory organs, as in the avian embryo, is pulmonary ventilation changing in parallel with metabolic rate? As mentioned earlier, this is the case in mammals since the lungs are the oruy possible route for gas exchange, thus pulmonary ventilation is forced to follow metabolic rate. What ifthe secondary gas exchanger is able to fully meet the metabolic requirements ofthe embryo? 9 Ifpulmonary ventilation followed total metabolic rate, then changes in metabolism would lead to equivalent changes in ventilation even though the requirements may already be met by a secondary source.
Multiple aspects must be considered before attempting to answer this question experimentally. Firstly, birds are essentially poikilothermic animaIs during their embryonic development and develop thermoregulatory mechanisms only after hatching
(Tazawa et al., 1989); hence, ambient temperature is the determinant ofthe embryo's body temperature. The latter is expected to directly influence metabolic rate.
Verification that the metabolism ofthe embryo could indeed be altered with changes in ambient temperature is essential to the viability ofthe model and needs to be tested 1.
Secondly, the period oftransition from diffusive to convective gas transport represents the time-window ofinterest to be investigated. The establishment and duration ofthis changeover also needs to be demonstrated by monitoring separately, but simultaneously, the gas exchange ofthe CAM and lungs as a function oftime and of ambient temperature.
Renee, these two issues, the effects oftemperature on the embryo's metabolic rate and the time course ofthe chorioallantoic and lung gas exchange, will be experimentally addressed first.
1 will then examine the metabolism-ventilation relationship in the chick embryo at term, taking advantage ofthis dual gas exchange model, to investigate the coupling between ventilation and total metabolic rate during changes in ambient temperature. In separate studies, 1 will explore, firstly, the possibility that the curled-up posture ofthe
1 The effects oftemperature on V02 throughout incubation are demonstrated in Appendix A. 10 embryo inside the egg may present a constraint to the embryo's ventilation and secondly, the extent ofventilatory chemosensitivity during the late phases of development. AlI three aspects allow for a deeper understanding and novel perception into the regulation ofbreathing in the avian embryo. Chapter 2
Metabolic Control ofPulmonary Ventilation in the Developing Chick Embryo
The material comprising this chapter has been accepted in Respiration Physiology and is presently in press. 12 2. Metabolic Control ofPulmonary Ventilation in the developing
chick embryo
2.1. Abstract
In birds, during the period from the breaking ofthe air cell by the beak (internaI pipping) to hatching, pulmonary ventilation CVE) begins and gas exchange is jointly provided by the lungs and the chorioallantoic membrane (CAM). We asked to what extent, during this phase oftwo concurrent gas exchange organs, changes in the embryo's metabolic needs were accompanied by changes in VE. The carbon dioxide and oxygen exchange rates (VC02, Vo2) through lungs and CAM were separately, but simultaneously, measured in chicken embryos at 20-21 days ofincubation, while VE was calculated from the measurements ofpressure oscillations in the air cell during breathing.
During the last 24 hours ofincubation, lung V02 and VC02 gradually increased as the corresponding CAM values declined. An increase in egg temperature from 33 to 39°C increased the embryo's total metabolic rate, especially when the lungs were the predominant gas exchange route. Whether metabolism increased because ofthe embryo's development or because ofthe increase in temperature, VE was linearly proportional to lung V02 and Vco 2, and not to the embryo's total metabolic rate. Rence, in the developing chick embryo, VE control mechanisms sense peripheral tissue requirements via the gaseous component ofcellular metabolism. 13 2.2. Introduction
The normal development of the avian embryo inside the egg is ensured by the gas exchange provided by the CAM (Wangensteen and Rahn, 1970/71). This highly vascular structure, in conjunction with the porosity of the eggshell, permits diffusion of oxygen and carbon dioxide between the environment and the blood (Tullett and Deeming, 1982), a function which could be paralleled to that of the placenta for the mammalian foetus.
During incubation, the continuaI loss of water through the membranes and shell favours the formation ofan air pocket ("air cell") at the blunted end ofthe egg. Toward the end of incubation, the embryo pierces the air cell membrane with its beak ("internaI pipping") and after a period, which in the chicken embryo is approximately 24 hours (Dawes, 1981;
Burton and Tullett, 1985), it begins to rupture the eggshell ("external pipping"). In the chick embryo breathing-like movements, and therefore the initiation of pulmonary air convection (VE), begin at the onset of, or even before, internaI pipping (EI-Ibiary et al,
1966; Vince and Tolhurst, 1975; Dawes, 1981). Hence, this is quite different from the mammalian case, in which \TE and placental diffusion overlap only at the time ofbirth for no more than a few minutes. In the avian embryo, during the last phases ofdevelopment,
\TE occurs in conjunction with two gas exchange organs operating together, the CAM and lungs.
After hatching, the embryo's gaseous metabolism is accomplished entirely by the lungs, at which time metabolic rate and \TE must be tightly coupled. In mammals, such a coupling has been demonstrated in many conditions, although the mechanisms responsible for it remain speculative (Mortola and Gautier, 1995). Ifcellular enzymatic 14 activities or the rates ofchange in sorne ofthe cellular energy substrates were important factors in dictating the \TE leve1, one may expect changes in the whole embryo's metabolic rate to be accompanied by corresponding changes in \TE. On the other hand, if only the gaseous component ofmetabolic rate (and therefore, the 02 and CO2 levels) was the important factor in controlling the level of\TE, it may be argued that during the period ofoverlap between CAM and lungs, \TE should be coupled only to that component of gaseous metabolism which is not exchanged by CAM. In other words, changes in \TE may be more closely related to pulmonary gas exchange than to total gas exchange. We tested this possibility in chicken embryos by simultaneously measuring oxygen consumption (Vo2) and carbon dioxide production (\TC02) through both CAM and lungs, together with YB, as the embryo's metabolism was raised by an increase in ambient temperature.
23.kfaœriahandkfdhods
FresWy laid fertilized eggs ofWhite Leghorn chickens were obtained from a local supplier. Eggs were weighed with a digital balance and placed in a still air incubator
(Hova-bator Model 1602), at 38°C and 60% relative humidity. Rotation to avoid adherence between the embryo and shell membranes was automatically performed four times per day. Day 0 was considered the first day in the incubator.
Measurements were performed starting on day 20, with a set up slightly different depending on whether or not external pipping had already occurred (figure 2.1). InternaI pipping was recognised by transillumination as a rupture ofthe air cell membrane, 15 whereas external pipping was recognized by the presence ofa hole in the eggshell. The staging ofthe embryo was irrelevant for the purpose ofthe experiments, but was important for deciding the appropriate set up for the recording oflung gas exchange and pulmonary ventilation (see below). At the end ofthe measurements, the egg was opened, the embryo was separated from yolk and membranes, and weighed.
2.3.1. Preparation
In order to measure separately the gas exchange occurring through the lungs and the CAM, we first identified, by transillurnination, the profile ofthe air cell membrane, which delirnited the air ceIl, and traced it onto the shell. The blunted end ofthe egg was forced through a hole prepared out ofa layer ofparaffin sealing film (Parafilm®). The parafilm was carefully positioned following this reference mark, to isolate the blunted end ofthe egg from the remaining part, and its edges were glued to the eggshell with surgical glue. The egg was then placed into a small cylindrical container, which was sealed by the parafilm. This egg container was placed into a second, larger container, also sealed at the top. Both containers had sorne water to maintain full water vapour saturation. Two polyethylene tubes, positioned at the opposite ends ofeach container, permitted gas sampling as weIl as a continuous flow ofair at the rate of220 mVrnin, under the control ofcalibrated flowmeters (Figure 2.1A). This preparation allowed for a complete separation ofthe gases transferred through CAM and lungs. However, the air cell membrane is a highly vascularized component ofCAM (Wangensteen and Weibel,
1982), yet contributing to the gas concentration ofthe air cell. Therefore, oxygen and carbon dioxide concentrations in the inner container underestimated the total gas 16 exchange through the CAM, whereas those in the larger container overestimated the gas exchange through the lungs. This was taken into account in the computation ofthe corresponding VOz and VCOz (see section 2.3.3.).
In the case ofembryos which had already broken the eggshell (external pipping), the set up was slightly modified (figure 2. lB). A small polyethylene mask was sealed on the eggshell hole with removable dental polyether material covering therefore, the beak and nostrils, and the tubes ofthe outer container used for the embryos with internaI pipping were connected directly to the mask. The whole setup was submerged into a temperature controlled water bath.
2.3.2. Temperature Equilibration
A clear way to properly dictate the temperature inside the egg is to allow it to equilibrate with the temperature in a controlled environment, for example in a water bath.
The time required for equilibration was monitored by inserting a thermocouple through a small hole made in the middle of the egg. Thus, by placing the water bath at a desired temperature, the time needed for the egg temperature to reach that equivalent temperature was determined.
Figure 2.2 indicates that it took -50 minutes to reach such an equilibrium when first bringing the egg temperature down from 38 to 30 oC and a similar time for every 3 oC increase, up to 42 oc. Hence, throughout all the experiments to be described, measurements were taken -90 minutes after preparation ofthe egg, or after a change in bath temperature, to ensure complete temperature equilibration. 17
2.3.3. Gaseous metabolism
The tubes ofthe individual compartments were sealed for 15-100 sec, depending upon the degree ofmaturation ofthe embryo and temperature. The short period of occlusion was to limit the change in 02 and C02 concentration to no more than 0.5%. At the end ofthe occlusion, the compartments were individually flushed with air at a
l constant flow of220 ml·min- . The bolus ofgas was analyzed for 02 and CO2 concentration by a calibrated polarographic 02 analyzer (Sable Systems International
Fox, Henderson, NV) and a CO2 infrared analyzer (Sable Systems CAIB, Henderson,
NV) arranged in series. The output ofthe analyzers was recorded on paper. 02 and C02 concentrations ofeach compartment were calculated ±rom the time integral ofthe gas concentration curves digitized from the records with a graphies tablet connected to a minicomputer. The rates ofoxygen consumption (V02) and ofcarbon dioxide production
(VC02) were calculated ±rom these concentrations, corrected for the water vapour pressure at the egg temperature, multiplied by the flow and finally by the reciprocal ofthe time during which the compartment was maintained sealed (Frappell et al., 1989;
Frappell and Mortola, 2000). Furthermore, we measured the gas exchange contribution ofthe air cell membrane in pre-pipping embryos, that is, just prior to any lung gas exchange, and found that it averaged 20% ofthe total CAM (i.e. air cell contribution!
(CAM + air cell contributions». For this reason, in all experiments, the measurements of
o gas exchange ofthe CAM compartment were increased by 20% in order to compute V
02CAM and VC02CAM, and the corresponding amount was subtracted from what was measured in the lung compartment for the computation OfV021ung and VC021ung. 18
2.3.4. Pulmonary Ventilation
A polyethylene tube was inserted into the air cell or the mask, depending upon the
type ofset up (Figure 2.1) and leaks were prevented with a seal. The other end ofthe tube
was connected to a pressure transducer (Validyne DP45, Northridge, CA), the signal of
which was amplified and recorded on paper. The breathing pattern was measured by an
adaptation ofthe barometric technique originally described by Drorbaugh and Fenn
(1955; Mortola and Frappell, 1998; see Appendix B). In this case, the egg itself,
connecting tube and transducer were equivalent to the Drorbaugh and Fenn's 'box', with the embryo breathing inside it. Egg temperature and water vapour pressure were known.
Average 'box' temperature and relative humidity ought to be intermediate between the
egg and the outside ambient temperature and relative humidity. Renee, their value was
estimated on the basis ofthe relative air volume located inside the bath (comprising the air cell, mask and portion ofthe tube) versus the air volume outside it (comprising the
external portion ofthe tube and the pressure transducer). The volume ofthe air cell was taken as 15% ofthe egg's weight on day 0 (Burton and Tullett, 1985; Tullett and
Deeming, 1982), whereas the volume ofthe other components were measured directly.
The volume calibration constant, K, ofthe 'box' was obtained by injecting a known volume (Vcal) and recording the corresponding change in pressure (Pcal); K= VcallPcal.
Renee, tidal volume (VI) was calculated from K, temperature and water vapour pressure ofthe embryo and the corresponding values ofthe 'box' (Drorbaugh and Fenn, 1955;
Mortola and Frappell, 1998). The P decay after a volume injection had a time constant of
1.15 sec, i. e. ~ 10 times longer than the inspiratory time, indicating that the conductance 19 ofthe pores ofthe eggshell was too low to appreciably alter the P generated by the embryo during breathing. Breathing rate (f, breaths/min) and VI (Ill) were measured with a graphies tablet connected to a minicomputer, in which VE (ml/min) was calculated from f.VI.
2.3.5. Protocol andData Analysis
Once the egg was prepared, 90 min were allowed for full temperature equilibration. In a tirst set ofmeasurements (N = 20) the same egg was maintained over time at a constant temperature, either 33 or 39 oC, for the purpose ofrecording the progression ofthe embryo's total V02 and V C02, and ofits lung and CAM components.
Then, the main experiment was conducted on a total of52 eggs, with simultaneous measurements ofCAM and lung V02 and V C02, and breathing pattern. The tirst measurements were always at egg temperature = 33 oc. Three or four measurements of gaseous metabolism were conducted, each intermingled with 30-60 sec recordings of breathing pattern. Egg temperature was then raised to 39 oC and after 90 min, the measurements were repeated. The results ofthe various trials at a given temperature were averaged. In the end, the egg was opened and the embryo examined to exclude obvious abnormalities, and weighed.
For the purpose ofplotting the data ofbreathing pattern against gaseous metabolism, aIl data (including V02 and VC02) were averaged according to the breathing rate, in bins of5 or 10 breaths/min. The respiratory rate was preferred over VI as the primary variable for binning because ofits wider range (see Results). Minute ventilation was calculated from the binned values ofVI and f Data are presented as group means 20 ± 1 SEM. Average results at 33 and 39°C were statistically compared by two-tailed paired t test. In the case ofthe breathing-metabolism relationships, linear regressions were fitted through the data points, and the resulting functions were statistically compared for significant differences between slopes or intercepts. In aIl cases a significant difference was taken at P<0.05.
2.4. Results
AlI eggs included in the study were ofsimilar weight at the onset ofincubation, on average 58.9 g (± 0.2). At the time ofthe measurements (between day 20 and 21) the embryos averaged 40.9 g (± 0.8).
Figure 2.3 shows the progression oflung and CAM gas exchange in two eggs in which seriaI measurements were obtained at the same temperature of39 oC. In these, as in aH the other embryos, the transition between CAM and lungs was a process ofvariable duration, often taking close to one fuH day. This process was slower at 33 oC than at
39 oC, and in sorne instances, complete hatching did not occur at 33 oC and the experiment was terminated.
Absolute V02 values ofCAM and lungs for aH eggs studied either 10ngitudinaHy at a constant egg temperature, or at the two temperatures are presented respectively in
Figures 2.4 and 2.5. In both figures, the data points from the embryos with internaI pipping are mostly grouped on the right-hand side; in fact, for these embryos, the average
V02lungwas only 0.06 ml/min (at 33 OC) and 0.09 ml/min (at 39 OC) (Table 2.1). The larger range ofdata in each plot is, therefore, provided mostly by embryos with external 21 pipping, which had an average \1021ung three ta four times higher (Table 2.1), due ta the important increase in embryo's \102 after external pipping (Visschedijk, 1968a; Tazawa and Rahn, 1987). Whether from the seriaI measurements during the last two days of incubation at a constant temperature (Figure 2.4), or from those in which the temperature was changed from 33 ta 39 oC (Figure 2.5), the results were consistent in indicating that at 33 oC, the total \102remained around the same value (~0.25-0.30 ml/min) during the whole process from the pre-pipping phase (when CAM contributed the whole embryo's
\102) to the external pipping phase (when \1021ung almost equalled total \102). On the other hand, \102 was higher at 39 oC than at 33 oC, and even more so the more advanced the stage ofdevelopment. The same pattern was obtained for \1C02. On average for the embryos before external pipping, total \102 increased from 0.26 to 0.34 ml/min, with a
QlO of2.2; a similar QlO (2.5) applied ta the temperature-response of\102 after external pipping (Table 2.2).
The absolute differences in total \102, \1021ung and \102CAM when egg temperature was increased from 33 to 39°C are presented in Figure 2.6, as a function ofthe \102CAM at
33°C. In the early phases, the lungs did not contribute to the temperature-induced increase in total \102 (Figure 2.6, bottom), which instead was entirely due to the increase in \102CAM(Figure 2.6, middle panel). The CAM contribution was ~0.05 ml/min and did not increase with the progression ofthe hatching process. On the contrary, after the external pipping, the contribution of\102CAMto the increase in total embryo's \102 was close to nil, and \102lungwas the sole component for the embryo's rise in \102. The 22 changes in total Vcoz, VCOzlung and VcozcAMwith the progression ofthe hatching
process or with changes in egg temperature were similar to those described for VÛ2.
Mean values ofthe breathing pattern for embryos before and after external
pipping are presented, respectively, in Table 2.1 and Table 2.2. In the internal pippers, with an increase in temperature, the breathing pattern (f, VI and VE) did not change
significantly, whereas aH parameters increased significantly after the external pipping. No
correlation or a negative correlation existed between VE and VOzCAM or VCOzCAM, whereas positive relationships were recognizable when VE was plotted against either the total, or only the lung component ofgaseous metabolism (Figure 2.7). However, two important differences emerged between these two relationships. First, the total metabolism-\TE relationships differed significantly between 33 and 39°C (Figure 2.7, left
panels), and, second, they presented an obvious intercept on the Voz- or VCOz axis.
Conversely, no difference could be documented between 33 and 39°C for the VOZlung-YB, or VCOzlung-\TE relationships; hence, these functions could be combined into a unique
1 function which had a small intercept on the VE-axis (0.7-1.1 ml.min- ) (Figure 2.7, right panels). Similar plots for fand VI (Figure 2.8) indicated that the changes in VE presented in the previous figure were mostly contributed by f, and much less by VI. As was the case for VE, so were the VOzlung-f and VÛ21ung-VT relationships indistinguishable between 33 and 39 oC (Figure 2.8, top right); the resulting combined functions formed an intercept on the Y-axis, at a f of21 breaths/min and a VI of- 51 Ill.
2.5. Discussion 23 The question at the core ofthis study was whether or not, in the chick embryo,
changes in metabolic rate were accompanied by proportional changes in VE. The answer
is a positive one, in that an increase in the embryo's total metabolic rate was indeed
accompanied by a rise in VT, f, and VE. Furthermore, it emerged that the increase in
breathing was better related to the increase in gas transfer through the lungs, rather than
to the embryo's overall metabolic rate. Before discussing these results, it seems
appropriate to comment on two aspects which were prerequisites for this work, the
temperature dependency ofmetabolic rate, and the occurrence ofbreathing activity
before hatching.
2.5.1. Temperature andEmbryo 's Metabolism
The total V02 ofthe embryos (CAM+lung) at 39 oC was similar to values
previously reported at similar incubation temperatures (e.g. Visschedijk, 1968a; Tazawa
and Rahn, 1987). In the chicken embryo, as in the embryos ofother avian species, the
homeothermic capacity develops only after hatching2 (Freeman 1964; Nair et al., 1983).
Renee, a direct correlation between egg temperature and the embryo's metabolic rate was
expected. The QlO values that we found (2.2-2.5) were near values previously reported in
embryos before or after pipping (Nair et al., 1983; Tazawa et al., 1989), and correspond to the effect oftemperature on the speed ofmetabolic reactions.
2 Indeed, no evidence has been found to indicate the presence ofthe thennogenic uncoupling protein (UCP) in various tissues ofthe chick embryo. Refer to Appendix C. 24 At the low egg temperature of33 oC, the embryo's \102 remained essentially constant throughout the pipping phases because the progressive increase in lung gas exchange was compensated by the dedine in the gas transfer through the CAM. Whether or not there is a causative link between these two processes is not known. The increase in egg temperature to 39°C resulted in only a marginal increase in CAM gas exchange, and eventually, even this small increase was no longer apparent with the progression ofthe hatching process. Indeed, from morphometric estimates ofthe CAM diffusing capacity, and the fact that they almost coincided with the functional measurements, it was conduded that the CAM is working at, or near, maximal capacity (Wangensteen and
Weibel, 1982). Also, the dedine in the allantois gas exchange, beginning at -15 hours before hatching, was previously inferred from seriaI measurements ofthe O2 and CO2 transfer from the eggshell (Visschedijk, 1968a). CAM O2 exchange is compromised to a greater extent than that ofCO2, owing to the fact that the CAM resistance to CO2flow is about halfthat to 02 (Piiper et al., 1980). AlI this indicates that any additional metabolic requirements by the embryo, induding those imposed by its own growth toward the end ofincubation, or by an increase in egg temperature, almost exdusively depend on what the lungs can provide. This explains the fact that in the late phases ofdevelopment, before pipping occurs, the embryo's \102 can be 02limited (Stock et al., 1983; Ar and
Rahn, 1985; Xu and Mortola, 1988).
2.5.2. Breathing Activity
Very few data have been reported on the breathing pattern ofavian embryos, and these were almost invariably confined to the rate ofbreathing movements. Pettit and 25 Whittow (1982a), by placing the egg ofexternally pipped embryos into a chamber and applying the barometric methodology ofDrorbaugh and Fenn (1955; see Appendix B), quantified VT; it averaged 90 /lI, while f averaged 87 breaths/min. The present vaIues at
39 oC (average VT and f, respectively, 74 /lI and 80 breaths/min) are in good agreement with those earlier measurements.
The relationship between VÜ2lung and breathing (Figure 2.8) had an intercept on the Y-axis, indicating that sorne breathing activity could occur in the absence of pulmonary gas exchange. Indeed, sorne respiratory-like movements can be detected before the piercing ofthe air cell membrane (Vince and Salter, 1967, Vince and Tolhust,
1975). Even after internaI pipping, when the embryo has access to the air ceIl, these breathing acts do no imply that the lungs are a functional gas exchange organ. In fact, aeration ofthe lungs, indicated by their capacity to float in water, may take a few hours after internaI pipping (EI-Ibiary et al., 1966; Vince and Tolhust, 1975). SeveraI factors could contribute in retarding an effective pulmonary gas exchange weIl beyond the onset ofbreathing movements. First, in birds, the clearance ofthe pulmonary fluid and lung aeration are likely to be a much slower process than in mammals, owing to the different mechanical characteristics ofthe respiratory system. The avian lungs hardly move during breathing and air is unidirectionally driven through them by the pumping action ofthe air sacs, an action which may be hindered by the posture ofthe embryo inside the egg (EI
Ihiary et al., 1966; to he discussed in Chapter 3). In addition, the large gradient, as high as
50 mm Hg, ofthe O2 partial pressure between the air cell and the arterialised blood
(Piiper et al., 1980; Pettit and Whittow, 1982b) suggests the presence offunctional 26 shunts; these have been demonstrated before pipping (Piiper et al, 1980), but could aIso
be present after the onset ofbreathing.
2.5.3. Breathing and Changes in Egg Temperature
Previous studies in pipped embryos have shown a breathing activity dependency
on temperature (Nair and Dawes, 1980; Dawes, 1981; Nair et aI., 1983). Similarly, in the
embryos with external pipping, as egg temperature increased from 33 to 39°C we
observed an increase in f, and with it an increase in VI. The most interesting finding was
that the increase in VE occurred in proportion to the pulmonary component ofV02 and
VC02, and not to the embryo's total gaseous metabolism. In fact, a unique relationship
existed between pulmonary gas exchange and \TE, whether the changes in \T021ung and
\TC021ung were occurring spontaneously with the progression ofincubation, or with an increase in egg temperature. In the external pippers, in which lung predominates over
CAM gas exchange, the average values ofVE/V021ung and VENC021ung were between 25
and 35. These values are slightly lower than the average resting values (~35-45) ofadult
and newborn mammals (Stahl, 1967; Frappell et al., 1992; Frappell and Baudinette, 1995,
Mortola, 2001), but comparable to those (~25) ofadult birds (Bouverot, 1978). Renee, it
seems that the relationship between lung air convection and gas exchange, which is central to the control ofbreathing, is fully established before the completion ofthe hatching process.
On the other hand, prior to external pipping, the rise in egg temperature was ineffective on \TE or on the breathing pattern. At this time, temperature had a significant effect on V021ung, but its average increase from 33 to 39 oC was small in comparison to 27 that ofthe external pippers (respectively, 0.03 versus 0.15 ml/min, refer to Tables 2.1
and 2.2). From internaI pipping until the breaking ofthe eggshell, the air cell has low O2
and high C02 contents (Pettit and Whittow, 1982b). Renee, a low \TE sensitivity in the
internaI pippers could serve the purpose oflimiting the energy losses associated with
breathing. On the other hand, a low \TE sensitivity to increased egg temperature at this
stage ofdevelopment could also be due to a low or absent chemoreceptor sensitivity and
Chapter 4 may support this possibility.
In conscious, resting adult sheep with extracorporeal circulation through a
membrane lung, \TE decreased as the rate ofextracorporeal gas transfer increased, and
eventually \TE ceased when the removal ofCO2 equalled the CO2 metabolically produced, with normal arterial blood gas levels (Phillipson et al., 1981). In lamb and goat fetuses with similar preparations, regular breathing did not initiate even ifdisconnected from the placenta so long as the membrane lung did not allow the endogenously produced CO2to rise above normal (Kuipers et al., 1992; 1997; Kozuma et al., 1999). The present study is
similar to the extent that CAM can be seen as agas exchanger outside the lungs. When the embryo's metabolic level exceeded what CAM could accomplish, \TE proportionately increased. As in those experiments in fetus and adult mammals, the nature ofthe link between metabolism and \TE is not known, but we cannot exclude a role played by the arterial partial pressure ofCO2 (paC02). The graduaI hypoxia and hypercapnia at the end ofincubation, owing to the mismatch between the embryo's growing metabolic rate and the CAM gas transfer capabilities, are considered key parameters in the timing ofthe hatching process (Visschedijk, 1968a,b,c; Pettit and Whittow, 1982b). Presumably,
PaC02 could also represent the \TE stimulus when total metabolic rate is increased by 28 changes in temperature, although the close proportionality between VE and lung V02 or
VC02 would imply a very high gain ofthe VE response to PaC02.
In conclusion, in the chick embryo, during the period between internaI pipping and hatching, ventilatory changes are c10sely correlated to pulmonary gas exchange, and not to total metabolic rate. This implies that, at least at this stage ofembryonic development, ventilatory control mechanisms are linked to peripheral cellular needs not via neural or humoral information, but via the gaseous component oftissue metabolism. 1',.',\'''''' A: internai pipping
.-e::-_ ---.c.- - ...... '-..
JI/ata buth
P,.~.\.\lIrt: B: external pipping
Water bath
Figure 2.1. Schema of the experimental setup during the last 24 hours of incubation of the chick embryo, before (A) or after (B) the breaking of the eggshell. The parafilm isolates the air cell from the blunted end of the egg, permitting the separation of gas transfer through the lungs ('pulmonary gas exchange') from that through the eggshell
('CAM gas exchange'). Insets show representative records of the pressure oscillations recorded as a result ofair convection through the lungs; time bar = 1 second. U 42 0 .... QI .. , ...... 39 ...... = =.. 36 QI eC- ...... QJ 33 ~ ~ QJ 30
o 10 20 30 40 50 60 time, min
Figure 2.2. Various egg temperatures plotted against time. With the use ofa temperature
controlled water bath, each egg was cooled down from 36 oC to 30 oC and then reheated to 33, 36, 39 and 42 oC (N = 10). SeriaI measurements were made every 5 minutes at
each temperature to assess the duration for egg temperature to equilibrate with water bath temperature. Dotted lines indicate the progression of each egg through the various temperatures. Bars are standard errors (when not visible, standard errors are embedded within the symbols). Lung 80 80
60
40
20
o1L----o._....L...----'_....1...... --'-----' L...-...... J...... J"-'-...... L...... L..o....J....J 0 o 2 4 6 8 o 2 4 6 8 ro U W ~ m 20 n Tuœ(hours) Tuœ(hours)
Figure 2.3. Changes in oxygen consumption through the Iungs and through the chorioallantoic membrane (CAM), expressed as a percent of the total, in two chick embryos at 39 oC, during the period between internaI pipping and hatching. Time 0 indicates the time of the tirst measurements. The transition between CAM and Iung gas exchange varied in time. 0.3
c'" o .-.... ~c ec.._e 0.2 fi:) C-=-- O e ~ '-' C 0.1 ~ l;>,() ~ o
0.0 L..---..I_--J._...... _....L._...... ---.;....L...-_&...... ---I_--Io_--L._...... _...L6..... 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Oxygen consumption, lung (ml/min)
Figure 2.4. üxygen consumption through the chorioallantoic membrane (VozCAM, Y- axis) plotted against the corresponding oxygen consumption through the lungs CVOzlung,
X-axis) in chick embryos over the time period between the onset ofinternaI pipping (low
VOzlung, high VozCAM) and hatching (high VOzlung, low VozCAM). Each embryo was studied at the same temperature, either 33 (closed circles) or 39 oC (open triangles), with seriaI measurements throughout the last two days of incubation. Oblique lines are the best-fit linear regression lines through the data points at each temperature. The dotted line joins values ofthe same total Voz (i.e. VOzlung + VOzCAM = constant) at 33 oc. ~ J:J.â 0.3 â â ~ â .... â â 0 .-=..-. .â â ...... C. ._=0.2 â e e â â ...... â r.f.l= â =0 -e ~ '-' âââ 0.1 â =CU • •• â el) • ~.• ~ -~ • â 0 .,• • â â 0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Oxygen consumption, lung (ml/min)
Figure 2.5. Oxygen consumption through the chorioallantoic membrane (V02CAM, y-
axis) plotted against the corresponding oxygen consumption through the lungs (V021ung,
X-axis) in chick embryos during the last 2 days of incubation. Each embryo was studied at two egg temperatures, 33 (c1osed circ1es) and 39 oC (open triangles). For other explanations, see Figure 2.4. Table 2.1. Average values ofgaseous metabolism and breathing pattern in chicken embryos with
internaI pipping
P
VOztotal (ml/min) [n=22] 0.26 (±0.01) 0.34 (±0.01) <0.00001
VCOztotal (ml/min) [n=22] 0.22 (±0.01) 0.30 (±0.02) <0.0001
VOzCAM (ml/min) [n=22] 0.20 (±0.01) 0.25 (± 0.01) <0.00001
VCOzCAM (ml/min) [n=22] 0.19 (±0.01) 0.25 (± 0.01) <0.00001
VOzlung (ml/min) [n=22] 0.06 (±0.01) 0.09 (± 0.01) <0.002
VCOzlung (ml/min) [n=22] 0.02 (±0.004) 0.06 (± 0.01) <0.05
VT (Jll) [n=21] 64 (±6) 64 (±5) ns
f(breaths/min) [n=21] 51 (±3) 51 (±5) ns
'.lE (ml/min) [0=21] 3.3 (±0.4) 3.4 (±0.5) ns
Values are means ± 1 SEM. P, level ofstatistical significance for the comparison between the
two temperatures (two-tailed paired t test); ns, not statistically significant (P>0.05). n, number
ofembryos studied. o 0.3 o o 00 o 0.2
0.1
0 00 .-= 0.0 ...... '" .-S -s 0.3 --~ ~ ..=~ 0.2 ~ A ~ 0.1 A ... AA A t "Cl A A A ~ U ~A AA. a "'" A A" 0.0 .. -.. A4t ...... • 4 ...... ~ il' A rf") A AA A A A V0 CAM A 2
U A Q Q'\ 0.3 A rf") A
A 0.2 A A ~ A
0.1 AA if>. A ~ ~ A 0.0 ~ A' • ·~AJ .A~' .
V02 1ung
0.00 0.05 0.10 0.15 0.20 0.25 CAM oxygen consumption at 33°C (ml/min)
Figure 2.6. Changes in oxygen consumption for the whole embryo
CV02total), through the chorioallantoic membrane (V02CAM) and the lungs
(V02Iung) when the egg temperature is increased ±rom 33 to 39 oc. Data
are plotted as a function of V02CAM at 33 oc. Dotted lines indicate no
change. Oblique continuous lines are the best-fit regression lines through the data points. Table 2.2. Average values ofgaseous metabolism and breathing pattern in chicken embryos with external pipping
P
Y02totai (mVmin) [n=30] 0.29 (±0.01) 0.44 (±0.02) <0.00001 ye02totai (ml/min) [n=30] 0.23 (±0.01) 0.36 (±0.01) <0.00001
Y02CAM (mVmin) [n=30] 0.10 (±0.01) 0.10 (± 0.01) ns
YC02CAM (mVmin) [n=30] 0.14 (±0.01) 0.16 (± 0.01) <0.005
Y02lung (mVmin) [n=30] 0.19 (±0.01) 0.34 (± 0.02) <0.00001
YC02lung (mVmin) [n=30] 0.09 (±0.01) 0.20 (± 0.01) <0.00001
VT (Ill) [n=22] 73 (±5) 74 (±4) ns f (breaths/min) [n=22] 59 (±3) 80 (±3) <0.00001
YE (ml/min) [n=22] 4.3 (±0.4) 6.0 (±0.5) <0.0002
Values are means ± 1 SEM. P, leve1 ofstatistical significance for the comparison between the twotemperatures (two-tailed paired t test); ns, not statistically significant (P>0.05). n, number ofembryos studied. ... 3r 2 2~/ 10 ... 1 10 /// 1 1 / 1 1 / 8 ... 1 1 / 8 ... 1 1 / !!. 1 1 / 6 1 1 / 6 ---= 1,1/ Os 1 / ...... 4 tyV// 4 Il ~ ... ! II/!!. 2 11/ 2 /1/ !!. "'... Q total CAM lung 0"= ..... 0 fi 0 ~ 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5 0"-..... Oxygen consumption (ml/min) =~ ~ 30/251 20/ 10 ... 1 1 / ... 1 1 / è 1 1 / ~ 1 1 / = 8 ... 1 1 / 8 § ... 1 1 / !!. 1 1 / - 6 1 1 / 6 ~= / fI// 4 ~/ 1 7 4 /11;t> ... 11/ !!. 2 11/ !!. "' ... total /1/ CAM lung 0 iJI 0 0.0 0.2 0.4 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5 Carbon dioxide production (ml/min)
Figure 2.70 Pulmonary ventilation (VE, Y-axis) plotted against oxygen consumption
(V02, X-axis, top panels) and carbon dioxide production (VC02, X-axis, bottom panels), in chick embryos at 33 oC (open symbols) and 39 oC ifilled symbols). Data are plotted against the gaseous metabolism ofthe whole embryo (left), the component exchanged by the chorioallantoic membrane (CAM, middle) or the component exchanged by the lungs (right). Values were binned according to breathing rate (see
Section 2.3. Materials and Methods and figure 2.8.) and the symbols represent the mean values ofeach bin. Dashed lines join constant VE/V02 or VEVC02 ofthe values indicated. Continuous lines are the linear regressions through the data points. total lung
.- 120 120 .~ 100 100 'VJ.c 10j ~ ,.Q 80 80 '-' QJ ... 60 60 E ~ .,. 40 40 ..c...= ~ 20 • • 20 ~ = 0 0 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5
100 100 • • • .- =t 80 80 -'-'
ë 60 60 0. Q= -;;;.. 40 40 -~ .,."=' E-! 20 20
0 0 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5 Oxygen consumption (ml / min)
Figure 2.8. Breathing rate (top, Y-axis) and tidal volume (bottom, Y-axis) plotted against oxygen consumption (X-axis) of the whole embryo (teft) or of the lungs only
(right), in chick embryos at 33 oC (open symbols) and 39 oC (jilled symbols). Data were binned according to breathing rate. Continuous lines are the linear regressions through the data points. Chapter 3
Respiratory Mechanics in Chick Embryos Before and After Exteriorization from the Eggshell
The material comprising this chapter has been submitted to: Journal ofExperimental Biology 30
3. Repiratory Mechanics in Chick Embryos Before and ACter
Exteriorization from the Eggshell
3.1. Abstract
In the chicken embryo, pulmonary ventilation (VE) and pulmonary gas exchange begin approximately one day before the completion ofhatching. We asked to what extent the posture inside the egg, and the presence ofthe eggshell and membranes, may alter the mechanical behaviour ofthe respiratory system. The passive mechanical properties ofthe respiratory system were studied in chicken embryos during the internaI pipping phase
(rupture ofthe air cell) or the external pipping phase (hole in the eggshell). Tracheal pressure, and changes in lung volume were recorded during mechanical ventilation, first, with the embryo curled up inside the egg, then again after exteriorization from the eggshell. In the internaI pippers, respiratory system compliance and expiratory resistance, respectively, increased and decreased after exteriorization, whereas the mean inspiratory impedance did not change. In the external pippers, exteriorization had no significant effects on either respiratory compliance, resistance, or impedance, and the values were similar to those ofnewly hatched chicks. We conclude that, in the chicken embryo, its membranes, the eggshell and the curled up posture do not represent a significant mechanical obstacle to VE during the last phases ofdevelopment. 31
3.2. Introduction
In birds, a large portion ofthe prenatal development depends on gas exchange through the chorioallantoic membrane (CAM), a highly vascular structure, which, together with the porosity ofthe eggshell, permits gas diffusion between the blood and the environment (Tullett and Deeming, 1982). Toward the end ofincubation, after the embryo's piercing ofthe air cell ("internaI pipping"), the lungs provide an additional route for gas exchange. In the chicken embryo, CAM and lungs operate concurrently for about one day before hatching is completed (Dawes, 1981; Burton and Tullett, 1985).
During this time, pulmonary gas exchange gradually prevails over CAM, especially after the breaking ofthe eggshell ("external pipping"), and this process is closely matched by the progressive establishment ofpulmonary ventilation (VE) (see Chapter 2).
Rence, differently from mammals, in which \TE and pulmonary gas exchange overlap with placental gas exchange only at birth and for less than a few minutes, in birds, \TE, pulmonary and CAM gas exchange occur together for many hours or even days before hatching. In mammals, mechanical factors, including the presence ofthe surrounding amniotic fluid and the stiffness ofthe maternaI uterine and abdominal tissues, may impede lung expansion ofthe fetus. In birds, the lungs represent rather rigid structures, which hardly move during breathing, while air is pumped through them unidirectionally by co-ordinated inflation and deflation ofvarious air sacs. It is the action ofthe latter that could be hindered by the posture ofthe embryo inside the egg (EI-Ibiary et al., 1966) and by the presence ofmembranes and eggshell. Ifthis was the case, the 32 impedance ofthe embryo's respiratory system should decrease after its exteriorization from the egg. In the following study, we have explored the passive mechanical properties ofthe respiratory system in chicken embryos during the last phases ofdevelopment, before and after the complete removal ofthe eggshell and membranes, hence, their full exteriorization.
3.3. Materials andMethods
FresWy laid fertilized eggs ofWhite Leghorn chickens were purchased from a local supplier. Eggs were incubated in standard air still incubators (Hova-bator Model
1602), at an ambient temperature of38°C and 60% relative humidity, with rotation four times per day to avoid membrane adhesion. Day 0 was considered the starting day of incubation. The progression ofembryonic development was established by transillumination. Experiments were conducted on embryos during the last two days of incubation (day 20-21) and in newly hatched chicks. Each group consisted of 12 animaIs separated as, firstly, embryos during the internaI pipping phase (i.e. after the beak tears the air cell membrane, recognised by transillumination), secondly, embryos during the external pipping phase (i.e. after rupture ofthe eggshell, recognised by a hole in the shell), and thirdly, hatcWings (within the first 12 hours offull hatching).
For the experiments on the embryos, a small region ofthe eggshell covering the air cell was opened, the wing covering the head was lifted and the head ofthe embryo was carefully exteriorised in order to expose the neck. The embryo was anaesthetised with an intramuscular injection ofSomnotol® in the wing muscles. The wing was then repositioned inside the egg. A small T-shaped tracheal cannula was inserted through an 33 incision in the upper portion ofthe extra-thoracic trachea, just below the syrinx. The cannula was directly connected to a small animal ventilator via a pneumotachograph
(Mortola and Noworaj, 1983), which was then connected to a differential pressure transducer for the measurement ofairflow. The airflow signal was amplified and, by electronic integration, it provided the changes in lung volume (V). A side arm ofthe cannula was connected to a calibrated pressure transducer for the measurement ofairway pressure (P). Both P and V were recorded on a two-channel pen recorder. The stroke volume ofthe ventilator was adjusted to deliver a peak-inflation pressure of5 cm H20, at a rate of20 cycles per minute. After at least 3 minutes ofmechanical ventilation to normalize the mechanical history ofthe lungs, three full ventilatory cycles were recorded at the paper speed of25 mm/sec (Figure 3.1).
The eggshell and membranes were carefully eut fully open until the embryo was completely exteriorised. The wings and legs were extended, and artificial ventilation resumed. Ifneeded, the stroke volume ofthe ventilator was adjusted to maintain the peak inflating pressure at 5 cm H20. After at least 3 minutes, three full cycles were recorded.
At the end ofthe measurements, the embryo was weighed on a digital scale. For comparative purposes, measurements were also obtained in newly hatched chicks, within
12 hours after hatching.
Respiratory system compliance (Crs, ml/cm H20) was computed from the change in V and Pat end-inflation, i.e. at zero flow, as Crs=ùV/ùP. The deflation time constant ofthe respiratory system (trs, sec) was computed from the exponential fitting ofthe V 34 decay during deflation. From -rrs and Crs, the expiratory resistance ofthe respiratory
l system (Rrs, cm H20 . ml- . sec) was then caIculated (Rrs = us/Crs).
l The mean inspiratory impedance ofthe respiratory system (Zrs, cm H20 . ml- • sec) was calculated as the ratio between the mean inspiratory tracheal pressure and the mean inspiratory flow rate. The mean inspiratory tracheal pressure was the total area underneath the tracheal P wave during inflation divided by the inflation time (TI); mean inspiratory flow rate was 8V over TI.
Group data are presented as means ± 1 SEM. Statistical comparisons ofaverage values before and after the exteriorization ofthe embryo (either internaI or external pippers) were done by two-tailed paired t tests. Comparisons among the three groups
(internaI pippers, external pippers and hatchlings) were performed by one-way ANOVA with post hoc Bonferroni limitations for the three inter-group comparisons. In aU cases, a significant difference was defined at P<û.ûS.
3.4. Results
Mean values ofthe variables measured in the embryos, inside and outside the egg, and in hatchlings are presented in Table 3.1.
3.4.1. Internai Pippers
Exteriorization from the egg resulted in an increase in Crs in 9 out of 12 embryos, with a mean increase of 18%, and a drop in expiratory Rrs in aU embryos, with a mean 35 drop of28% (Figure 3.2). As a result ofthese changes the expiratory 'us was -13% shorter. The mean inspiratory Zrs was not significantly modified.
3.4.2. External Pippers andHatchlings
At this stage ofdevelopment, exteriorization ofthe embryo did not result in a significant change ofany ofthe parameters measured (Table 3.1).
After hatching, none ofthe mean values pertinent to the mechanical properties of the respiratory system differed significantly from those measured in the exteriorized embryos (Table 3.1). The oruy exception was Crs/kg, which in the hatchlings significantly exceeded the value ofthe external pippers (Figure 3.3).
3.5. Discussion
The results ofthis study indicated that exteriorization ofthe embryo resulted in sorne changes in the parameters ofrespiratory mechanics oruy in the internaI pippers, with no effects in the embryos after external pipping. It could be argued that the lack ofa significant difference might have been the effect ofthe rather large variability. However, the similarity ofthe mean values before and after exteriorization in the external pippers, the possibility ofdetecting differences when they occurred (for example, Crs and Rrs in the internaI pippers) and the fact that each animal acted as its own control, are aH arguments against the possibility ofa Type II statistical error.
3.5.1. Comparison with Mammals andAdult Birds 36
ln the domestic fowl, as in other birds (Scheid and Piiper, 1969; Barnas et al.,
1991, also for additional references), Crs, on a per kg basis, is several times higher than in mammals. The present values of5-6 ml·kg-1·cm R20 are at the lower end ofvalues of adult birds, and about four times the values ofnewborn (Figure 3.4, top) or adult mammals. Renee, the birds' higher Crs is a characteristic difference from mammals already apparent in the perinatal period.
The Rrs values are more difficult to compare among animaIs ofdifferent size or age, because Rrs does not bear a direct relationship to body weight. Furthermore, resistance measurements can vary greatly even with seemingly small differences in methodology. In birds, the few measurements ofRrs during artificial ventilation with patterns similar to that adopted here (rather large VI and small rates) gave values ofRrs similar to (Kampe and Crawford, 1973) or higher (Barnas et al., 1991) than expected for mammals ofcomparable body size. We measured values substantially higher than those measured in newborn mammals ofsimilar size (Figure 3.4, bottom). The small dimensions ofthe airways and the possibility ofsorne remnant pulmonary fluid could explain the high Rrs values. It is also possible that the air sacs themselves, once inflated, may restrict neighbouring air pathways. Indeed, on sorne occasions we noticed progressive overinflation, especially in the abdominal regions, which was then resolved by a slight repositioning ofthe animal.
During spontaneous breathing in the awake condition, a combination offactors including a shallow and rapid breathing pattern and neurochemical control ofairway resistance (Molony, 1978; Barnas et al., 1991), can lower Rrs. It has been pointed out that the high Crs coupled to a high Rrs implies that, in birds, us is much longer than in 37 mammals (Brackenbury, 1973). Similarly, we found extremely long "CrS in both embryos and hatchlings. However, it should be emphasized that our measurements were obtained during mechanical ventilation in passive conditions. In active (awake) conditions, that is, when the respiratory muscles generate the pressures for breathing, it is likely that the respiratory system would behave functionally as ifit was less compliant than apparent from the passive measurements, thus shortening its effective trs. In newborn mammals, in active conditions, Crs can be halfthe passive value (Mortola, 2001). In adult birds, contraction ofthe expiratory muscles is a necessity to accelerate the deflation phase of the breathing cycle; this is likely to be the case also in the chicks, but no data on respiratory muscle activity in hatchlings have ever been produced
3.5.2. InternaI Pippers
In the internaI pippers, freeing the embryo from the membranes and eggshell resulted in an increase in Crs and a drop in expiratory Rrs, with insignificant changes in inspiratory Zrs. Because the inflations were performed at very low rates, the inertance
(acceleration dependent) component ofthe total inflation pressure must have been negligible. Hence, the fact that, with exteriorization, the increase in Crs was not accompanied by a drop in Zrs indicates that the pressure to overcome the inspiratory resistance (i.e. airflow-related) was the component ofoverwhelming importance. In birds, differently from mammals, resistance can be higher in inspiration than in expiration
(Brackenbury, 1972). During the internaI pipping phase, CAM gas exchange is still an important, and possibly predominant, route ofgas exchange (see Table 2.1, Chapter 2), 38 and the air sacs are likely to contain fluid. Presumably, exteriorization contributed to the redistribution ofembryonic pulmonary fluid, favouring the expansion ofthe air sacs, hence increasing Crs.
3.5.3. External Pippers
At this later stage ofdevelopment, \TE is fully established and pulmonary gas exchange is the primary means ofgas exchange (Menna and Mortola, 2001). Therefore, we were particularly interested in the effects ofexteriorization on respiratory mechanics at this stage ofdevelopment because ofthe visual impression, as previously pointed out
(EI-Ibiary et al., 1966), that the curled posture inside the egg could limit the expansion of the air sacs. In snakes, which have an elongated air sac extending a large portion oftheir body, the coiled position causes a significant reduction ofCrs in comparison to the straight position (Bartlett et al., 1986). This was not the case for the embryos in the external pipping phase, indicating that whatever the limitation in the degrees offreedom tOOt the curled position inside the egg introduced, it had no detectable mechanical implications. Our measurements inside the egg were performed with the blunted end of the eggshell partially open. The question arises, however, as to what could be the implications on respiratory mechanics ofbreathing inside the completely sealed egg.
3.5.4. The SealedEgg
During avian incubation, the water loss due to the porosity ofthe eggshell contributes to the formation ofan air pocket. From the difference in egg weight between the onset and the termination ofincubation, and knowing that the embryo's body density 39 is 1.025 (results not shown), one can calculate that the volume ofgas inside the egg at end-incubation is 8-9 ml. This volume correspondingly dictates the size ofthe air cell
(Tullett and Deeming, 1982; Burton and Tullett, 1985). The presence ofthis compressible air space creates a situation mechanically very different from that ofthe mammalian fetus, which finds itselfin a totally liquid filled, and therefore incompressible, environment. After piercing the air cell membrane, the embryo's breathing shifts gas between the air cell and the respiratory system, with no change in the total air volume of the egg (neglecting the air decompression introduced by airflow resistance). After external pipping, with an important rupture ofthe eggshell, the airspace ofthe egg is in communication with the outside environment; hence, no body-surface pressure can be generated as the result ofbreathing. Oruy during the early phases ofthe external pipping phase, in the unique event that the beak protrudes with a tight fit through a small hole in the eggshell, does the possibility exist for a build up in pressure during inspiration, which could oppose the expansion ofthe air sacs. In such a case, assuming an air cell volume of
9 ml and the average VT of0.08 ml (Pettit and Whittow, 1982; Menna and Mortola,
2001), this would result in a positive pressure around the embryo of9/(9-0.08)=0.9% of 1 atm, or 9 cm H20. This would be an important elastic load that could substantially reduce the effective Crs and compromise the embryo's VE. The fact that no systematic differences in VT were observed among embryos at various phases ofthe hatching process (refer to Tables 2.1 and 2.2, Chapter 2) indicates that this possibility is a rather remote eventuality.
In conclusion, the measurements performed here indicated that respiratory mechanics was slightly affected by the posture inside the egg in the internaI pippers, 40 whereas no effects were detectable in the external pippers. Rence, there is no indication that the curled position ofthe embryo within the egg presents a limit to VE in the late phases ofembryonic development. 1 ml
1 sec
Figure 3.1. Records of volume and tracheal pressure during
artificial ventilation. Respiratory system compliance (Crs) was
measured from the change in volume (8V) and the corresponding
pressure change (8P), Crs =8V/ùP. The V decay during deflation
was fitted by an exponential function for the calculation of the
respiratory time constant (us), from which the resistance (Rrs)
was calculated as us/Crs. Mean inspiratory impedance (Zrs) was
calculated as the ratio of the mean value of tracheal pressure
during inflation and the mean inspiratory flow rate. Table 3.1. Mechanical properties ofthe respiratory system inside and outside the egg
inside outside outside/inside
InternaI pippers (BW 42.1 g ± 0.3, n=12):
Compliance 0.26 ± 0.02 0.30 ± 0.02 • 1.18 ± 0.10
Mean Expiratory Resistance 3.17 ± 0.32 2.16 ± 0.20· 0.72 ±0.06
Expiratory time constant 0.83 ± 0.09 0.64 ± 0.07· 0.87 ±0.12
Mean Inspiratory impedance 3.79 ± 0.48 3.48 ± 0.47 0.92 ± 0.06
External pippers (BW 43.2 g ± 0.5, n=12):
Compliance 0.23 ± 0.01 0.24 ± 0.03 1.04 ± 0.08
Mean Expiratory Resistance 2.80 ± 0.33 2.54 ± 0.24 0.96 ± 0.07
Expiratory time constant 0.66 ± 0.10 0.59 ± 0.07 0.95 ± 0.07
Mean Inspiratory impedance 3.86±0.19 4.12 ± 0.25 1.09 ± 0.07
Hatchlings (BW 42.5 g ± 0.7, n=12):
Compliance 0.32 ± 0.03
Mean expiratory resistance 2.39 ± 0.22
Expiratory time constant 0.79 ± 0.13
Mean inspiratory impedance 3.81 ± 0.12
Values are means ± 1 SEM. Compliance, ml/cm H20. Resistance and impedance, cm H20 . rI • sec. Time constant, sec. • significant difference between inside and outside (paired t test, P<0.05).
BW, body weight. n, number ofembryos studied. -0 M 0.4 ==e ::::.C.J e 0.3 ~ --C.J .-=~ -~ Q.; -..= - 0.2 rIJ e ~ Q ~ U ~ ..=~ ...... 0.2 0.3 0.4 ~ .-"C rIJ ~ ...... 1 -C.J ~ 5000 0= ~ ~ 1- ~ 4000 ==e C.J 3000 ~ --C.J .....=~ ~ 2000 .-~ ~ ~
2000 3000 4000 5000 Inside the eggshell
Figure 3.2. Respiratory system compliance (top panel) and resistance (bottom panel) in chick embryos with internaI pipping, inside (X-axis) and outside (Y-axis) the eggshell. Oblique lines are identity lines. In these embryos, exteriorization resulted in an increase in compliance and a reduction in resistance. 8 :'
0M ==l3 Col 6 "7 ~ ~ Ë 4 -Q) ~ .-=œ -c.. 2 El u0 o
160
Col ~ 140 ~ ~ ..... 120 '- o 100 ==M l3 80 Col
--~ 60 =œ 1;; 40 .,.. ~ ~ 20
o internai external hatchlings pipping pipping
Figure 3.3. Average values ofthe parameters ofrespiratory mechanics measured in chicken embryos before and after exteriorization from the eggshell and in hatchlings. Columns represent mean values (n = 12), bare are standard errors. *, significant difference (P =M chick embryos { ~ e and hatchlings Q ~ 0.1 -e ~ newborn eatberians 0.01 10 100 .- 10000 ~ QJ ~ - ~} chick embryos --- and hatchlings -0 =M 1000 e ~ newborn eutherians /' guinea pig • cat. Resistance 100 10 100 Body weight, g Figure 3.4. Average values of respiratory system compliance (top) and resistance (bottom) in the chick embryos and hatchlings and in sorne newbom mammalian species, plotted at the corresponding body weight. The continuous lines refer to the allometric curves of newbom eutherian mammals (from Mortola, 2001). Filled triangles, embryos inside the eggs. Open circ1es, embryos and hatchlings outside the eggs. Chapter 4 Ventilatory Chemosensitivity in the Chick Embryo The material comprising this chapter has been submitted to: Respiration Physiology 42 4. Ventilatory Chemosensitivity in the Chick Embryo 4.1. Abstract In the avian embryo toward the end ofincubation pulmonary ventilation (VE) provides an important route for gas exchange, in addition to what is normally occurring through the chorioallantoic membrane (CAM). We asked to what extent changes in carbon dioxide and oxygenation would modify VE in the externally pipped chicken embryos. Hypercapnia (5% CO2, in normoxia) and hyperoxia (100% O2) resulted in, respectively, a minor drop (-6%) or no effect in total (ie. CAM + lung) oxygen consumption (V02), whereas in hypoxia (10% O2) total V02 halved. Furthermore, the V02 occurring through the lungs alone (V021ung) was measured along with VE, as the respiratory gases through the CAM compartment were changed. Hyperoxia had no effects on VE and slightly reduced V021ung, whereas hypoxia resulted in sorne (+14%) hyperpnea. The latter was entirely due to an increase in tidal volume (VT) and perfectly matched the increase (+14%) OfV021ung. Hypercapnia did not change V021ung; however, it did increase VE (+26%) again, through an increase in VT. Thus, we conclude that the chicken embryo at term presents a clear VE-sensitivity to CO2, but seems to have a very weak VE-sensitivity to changes in arterial oxygenation. In addition, the results are compatible with the view that, in the chicken embryo, the level ofVE is sensitive to V021ung, i.e. to that component oftotal gaseous metabolism that is not exchanged through the CAM. 43 4.2. Introduction In mammals, breathing activity can already be recognized during the fetal period, although gas exchange is solely provided by the placenta with no contribution from the lungs (Jansen and Chernick 1991; Mortola, 2001). Similarly in birds, respiratory activity begins well before hatching (El-Ibiary et al., 1966; Vince and Salter, 1967; Vince and Tolhurst, 1975; Dawes, 1981). Nonetheless, for the majority ofincubation, gas exchange is provided exclusively by the chorioallantoic membrane (CAM). As mentioned before, the CAM is a highly vascular structure lining the inner surface ofthe egg, which in conjunction with the porosity ofthe eggshell, permits diffusion ofoxygen (02) and carbon dioxide (C02) between the environment and the blood (Wangensteen and Rahn, 1970/71; Tullett and Deeming, 1982). With the progression ofincubation, the continuaI loss ofwater through the membranes and shell favours the formation ofan air cell at the blunted end ofthe egg. Thus, when the embryo pierces through the air cell membrane with its beak (internaI pipping), it gains access to air and pulmonary ventilation (VE) becomes an additional route for gas exchange. The importance ofpulmonary gas exchange increases further with the onset ofexternal pipping (dictated by a rupture ofthe eggshell), while the role ofCAM continues to decline until hatching is completed. Renee, differently from mammals, for a period, which in the chicken embryo is approximately 24 hours (Dawes, 1981; Burton and Tullett, 1985; Menna and Mortola, 2001), two gas exchange organs operate concurrently, the CAM and lungs. 44 Given the role ofYE in avian embryonic gas exchange, one may expect that the mechanisms ofYE chemosensitivity could be functional before hatching. In the mammalian fetus, hypercapnia stimulates respiratory-like activity, whereas hypoxia has no or depressant effects ( see Mortola 2001 for review). In the avian embryo, Dawes (1975) reported that the pressure oscillations obtained from the air cell ofintemally pipped eggs increased in amplitude when the egg was exposed to CO2. Very few studies have attempted measurements ofYE in chick embryos (Pettit and Whittow, 1982; Menna and Mortola, 2001), while hypoxic and hypercapnic ventilatory responses have never, to our knowledge, been recorded. The primary goal ofthis study was therefore to investigate the YE response to hypercapnia and to changes in O2in the extemally pipped chick embryo, following the hypothesis that YE chemosensitivity is already apparent at this stage ofdevelopment. Changes in gaseous composition can modify the metabolic level ofmany animais, including the chick embryo (H0iby et al., 1983; Stock et al., 1983, 1985; Tazawa et al., 1992). Furthermore, the metabolic level can influence the magnitude ofthe YE response (Mortola and Gautier, 1995; Mortola, 1996) and, in the chick embryo, YE seems particularly linked to that component ofoxygen consumption (Yo2) not exchanged through the CAM (see a/sa Chapter 2; Menna and Mortola, 2001). Hence, for a better interpretation ofthe YE response to changes in respiratory gases, both total YÛ2 and that component OfY02 provided by the pulmonary route (Y021ung) have been measured. 4.3. Materials and Methods 45 FresWy laid fertilized eggs ofWhite Leghorn chickens were obtained from a local supplier and were placed in a still air incubator (Hova-bator Model 1602), at 38 oC and 60% relative humidity. Rotation to avoid adherence between embryo and membranes was automatically performed four times per day. Day 0 was established as the first day in the incubator. Measurements were performed on externally pipped embryos (which occurred throughout day 20 and 21 ofincubation). This stage was recognized by the presence ofa hole in the eggshell. Two types ofexperiments were conducted on separate sets ofeggs. First, measurements oftotal gas exchange were obtained in air and after a briefexposure to an altered gas composition. Second, measurements ofgas exchange through solely the lungs, together with VE, were acquired while manipulating the gas composition flushed through the CAM compartment. 4.3.1. Total Gaseous Metabolism Total oxygen consumption (V02total) and carbon dioxide production (VC02total) of the whole egg (i.e. the gas exchanged through the lungs and CAM combined) were measured using the open-flow method (Frappell et al., 1992). The eggs were studied in sets oftwo. They were placed in a 1100 ml respirometer with the ambient temperature, measured by two thermocouples each placed at opposite ends ofeach other, preset at 39°C by a circulating water bath. The flow ofgas, either air or premixed gases from pressurized tanks, through the respirometer was controlled by a precision flowmeter set at l 950 ml·min- . Time was allowed for temperature and gas equilibration to take place. 46 Inflowing and outflowing gases were sampled, passed through a drying column (Drierite) and monitored by a calibrated polarographic 02 analyzer (aM-lI, Beckman) and by an infrared CO2 analyzer (CD-3A, Applied Electrochemistry), arranged in series. Gas concentrations were displayed on a computer monitor during on-line acquisition and V02 and VC02 were calculated from the product ofthe flow rate and the inflow-outflow concentration difference, in ml, at standard temperature, pressure and dry conditions (1 ml 02 STPD = 0.0446 mmol 02), averaged over several minutes. At the end, the eggs were opened and the measurements were normalized by the body weight ofthe embryos in kg. Data were collected on a total of 15 sets ofeggs (5 sets per group) during exposure to air and to either hypoxia (10% O2), hyperoxia (100% 02) or hypercapnia (5% CO2 in normoxia). Thus, each set ofeggs was exposed to no more than one gas mixture and the exposures lasted approximately 25 minutes, the last 5 minutes ofwhich were used for the calculation ofV02totai and VC02total. 4.3.2. VEandV021ung For these measurements, we adopted a setup similar to that previously used to separately measure the gas exchange ofthe lungs from that ofthe CAM in embryos during the external pipping phase (see Chapter 2, figure 2.1; Menna and Mortola, 2001). Briefly, the profile ofthe air cell membrane was identified by transillumination and was traced onto the shell. This delimited the boundaries ofthe two compartments. The blunted end ofthe egg was pushed through a hole prepared out ofa layer ofparaffin sealing film (Parafilm@). The parafilm was carefully positioned following the trace 47 around the shell, hence isolating the air cell from the remaining part ofthe egg, and its edges were glued to the eggshell with surgical glue. The egg was then placed into a small cylindrical container containing sorne water to maintain full water vapour saturation. The container was then sealed by the parafilm, which in turn enclosed the CAM compartment. Next, this egg container was placed into a second, larger container, also sealed at the top. Two polyethylene tubes, located at the opposite ends ofthe inner container, permitted the l delivery ofthe desired gases to the CAM compartment, at the rate of220 ml'min- , under the control ofa calibrated flowmeter. The measurements of\TE were conducted by placing a small polyethylene mask over the area ofthe eggshell that was cracked, that is, the area through which the embryo was pipping. This little mask, sealed in place with removable dental polyether material, therefore covered the beak and nostrils. Three polyethylene tubes trom the larger container were directly connected to the mask; two ofthem were for the measurements of pulmonary gas exchange and the remaining one was for monitoring \TE. The whole set up was submerged into a temperature controlled water bath, which was maintained at the constant temperature of39 oc. For more details, refer to Chapter 2, section 2.3.1. For the assessment of\T021ung, the tubes ofthe mask were sealed for no more than 30 seconds to ensure that the occlusion would not change the 02 and CO2 concentration by more than 0.5%. The tubes were then re-opened, thus allowing the mask to be flushed l with a constant flow of220 ml'min- . The gas was analyzed for O2 and CO2 concentration by calibrated 02 and C02 analyzers, arranged in series. The output ofthe analyzers was recorded on paper. O2 and CO2 concentrations were calculated trom the timeintegral of 48 the gas concentration curves digitized from the records with a graphies tablet connected to a minicomputer. V021ung and the rate ofC02 production (VC021ung) were calculated from the concentrations, corrected for the water vapour pressure at 39 oC, multiplied by the flow and finally by the reciprocal ofthe time during which the mask was sealed (Frappell et al., 1989; Frappell and Mortola, 2000). For the measurements OfVE, the third polyethylene tube ofthe mask was connected to a pressure transducer (Validyne DP45, Northridge, CA), the signal was amplified and recorded on paper. The breathing pattern was measured using the same technique described for the external pippers in Chapter 2, section 2.3.4. (Menna and Mortola, 2001). To summarize, it is an adaptation ofthe barometric technique (Drorbaugh and Fenn, 1955) where, in this case, the egg itself, connecting tube and transducer are equivalent to the Drorbaugh and Fenn's "box", with the embryo breathing l inside it. Breathing rate (f, breaths . min- ) and tidal volume (VI) were measured with a l graphies tablet connected to a minicomputer, in which VE (ml'min- ) was calculated from fVI. 4.3.3. Protocol andData Analysis Once the egg was prepared, 90 minutes were allowed for full temperature equilibration (Chapter 2, section 2.3.2.) as the containers (i.e. the separated CAM and lung compartments) were continuously flushed with air. Three offour measurements of lung gaseous metabolism were conducted, each intermingled with 15-30 second recordings ofbreathing pattern. The results ofthe various trials were averaged. After the measurements in air, the gas mixture flushed through the CAM compartment was 49 changed to either hypoxia (10% O2, N = 20), hyperoxia (100% O2, N = 20) or hypercapnia (5% CO2 in normoxia, N = 16), by connecting the inner container to a gas Impermeable bag which contained the desired gas mixture. After a period of 15 minutes, data ofpulmonary gaseous metabolism and VE were collected again. In the end, the egg was opened and the embryo was examined to exc1ude obvious abnormalities and weighed. Data are presented as group means ± 1 SEM. Average results ofthe responses to changes in gas mixtures were statistically compared by two-tailed paired t test. In aIl cases, a significant difference was taken at P<0.05. 4.4. Results 4.4.1. Total Metabolism Table 4.1 presents the mean values ofVû2totai in the various experimental conditions. In hyperoxia, gaseous metabolism hardly changed, whereas in hypoxia and hypercapnia, it dropped significantly, respectively to 49% (± 7) and 94% (± 2) ofthe corresponding air values. 4.4.2. Pulmonary Responses to Hypercapnia Hypercapnic gas de1ivered to the CAM compartment had insignificant effects on VÛ21ung, whereas it acted as an important stimulus on VE, which increased by 26% (Figure 4.1.). This increase was entirely due to the increase in VI (+21%), while fvaried 50 minimally. As a result ofthe hyperpnea, both VE/V0 2Iung and VE/VC0 2lung increased , respectively by 41 % and 21 %. 4.4.3. Pulmonary Responses to Changes in Oxygen Hyperoxia resulted in a small, but significant decrease in V0 2lung (-11. 5%) with no effect on VE or on the breathing pattern (Figure 4.2.) Conversely, hypoxia resulted in a slight increase in both V0 2lung and VE; because both variab.les increased by the same amount (+14%), VE/V02lung remained at the normoxic level. Changes in oxygenation had no significant effects on VC0 2lung nor on VE/VC0 2Iung. As was the case during the hypercapnic conditions, the observed hyperpnea during hypoxia was also solely determined by an increase in VI (+10%), with no significant changes in f (Figure 4.3.). 4.5. Discussion 4.5.1. Effects ofChanges in Oxygenation on Metabolism The hypometabolic effects ofhypoxia are a common finding in animaIs ofall classes and ages, and in avian embryos, the effects have been consistently observed throughout incubation (e.g. Ar et al., 1991; Tazawa et al., 1992). Tazawa et al. (1992) reported that V02 almost halved in externally pipped embryos during 10% 02, similarly to what we found. As well, they also observed sorne residual hypermetabolic effects during hyperoxia, although small (+8%), compared to the + 18% that they measured on day 16 or 18 ofincubation. One interpretation ofthe hypometabolic response is that the 51 metabolic rate ofthe growing embryo is close to, or even exceeds, the gas exchange capacity ofthe CAM, creating a situation of02-limitation (lWiby et al., 1983; Stock et al., 1983, 1985; Xu and Mortola, 1988; Tazawa et al., 1992). Hence, exposure ofthe egg to hypoxia may aggravate the limitation, lowering vo2, whereas hyperoxia may resolve it, increasing Vo2. We did observe the former, but not the latter phenomenon in the externally pipped embryos ofthe present study. It is possible that at the end of incubation, during normoxic breathing, the convection and gas exchange properties ofthe lungs may be effective enough to overcome the inherent 02-limitation ofthe embryo. When hyperoxia or hypoxia was applied to the CAM compartment alone, the oxygen exchange through the lungs responded as it may have been expected to based on gas diffusion. Because V02totai did not increase during hyperoxia, the partial pressure of oxygen (P02) should have increased both in the arterial blood and slightly in the venous blood, by a magnitude depending on the characteristics ofthe hemoglobin dissociation curve. This increase, by reducing the alveolar-venous gradient, tended to lower V02lung. Exposure ofthe CAM to hypoxia, on the other hand, increased the alveolar-venous P02 gradient, therefore raising the V02lung. Since, with the changes in oxygenation, the concentration ofC02 in the CAM compartment did not change, VCÛ2lung changed less than V02lung did. 4.5.2. Ventilatary Respanses ta Changes in Oxygen During hyperoxia in a few-day old mammals, as in adults, VE initially decreases and after a few minutes, it returns to, or even exceeds, the normoxic value (Mortola, 52 2001). The first acute response is believed to reflect a sudden silencing ofthe peripheral chemoreceptors. Thereafier, the brain tissue accumulation ofCO2, possibly due to the hyperoxic cerebral vasoconstriction and the reduction in hemoglobin capacity for CO2 (Raldane effect), stimulates breathing. Furthermore, in newbom infants, the hyperoxic increase in V02 contributes to the observed rise in VE (Mortola et al., 1992). In birds, as in mammals, the carotid bodies are the primary organ responsible for the VE response to hypoxia, and in the chick embryo at term, the carotid bodies resemble, both macroscopically and histologically, those ofthe adult (Murillo-Ferrol, 1967). In adult birds, the carotid chemoreceptors are functional during resting normoxic breathing. Rence, VE not only increases in hypoxia but also decreases with acute hyperoxia (Jones and Purves, 1970; Bouverot, 1978). The domestic fowl, however, may represent an exception to this behaviour because despite having active chemoreceptors (Bouverot and Leitner, 1972), the VE responses to hypoxia or hyperoxia have been found to be present in one study (Bouverot and Leitner, 1970), but reduced (hypoxia) or absent (hyperoxia) in another study (Brackenbury et al., 1982). Therefore, we cannot exclude that the present findings ofan absent Vl? response to hyperoxia and a very limited response to hypoxia in the chick embryo may represent a characteristic ofthis species, rather than a general feature ofthe avian embryonic phase. In addition, the hypoxic VE response was entirely due to an increase in VT, rather than to the more typical increase in breathing frequency (Mortola, 1996). This raises the possibility that even the small amount ofhyperpnea observed during the hypoxic conditions may not have been the effect ofhypoxic chemoreceptor stimulation. In a 53 previous study (see Chapter 2), we found that, in internally and externally pipped chick embryos, VE was linearly proportional to changes in pulmonary V02 and VC02' and not to the embryo's total metabolic rate. The present results are consistent with that view, in that the percent changes in VE during hypoxia or hyperoxia were very close to those of pulmonary, and not total gas exchange. Presumably, in the chick embryo, that component ofgaseous metabolism not exchanged through the CAM plays a pivotaI role in setting the levelofVE. 4.5.3. Respanse ta Hypercapnia In birds, differently from mammals, the VE response to C02 results from the activation ofnot only the central and peripheral chemoreceptors, but also from the intrapulmonary chemoreceptors. However, the relative contribution ofeach group of receptors remains controversial (Scheid and Piiper, 1986). In the chicken, both intrapulmonary and systemic (arterial and central) chemoreceptors participate in the YB response to C02 (Osborne et al., 1977). Previous measurements in chick embryos toward the end ofincubation indicated that the pressure oscillations in the air cell, presumably related to tidal volume, increased in amplitude when the whole egg was exposed to 5% CO2 (Dawes, 1975). Because the rate ofthe oscillations did not change, those results would indicatethat the YB response was exclusively mediated by tidal volume. Observations made many years age and limited to the frequency ofrespiratory-like movements, indicated minimal changes when chick embryos were exposed to CO2 (Windle et al., 1938; Romijn, 1948). AlI these previous observations are interestingly 54 consistent with the present results showing a hypercapnie \TE response mediated exclusive1y by an increase in VT. This is a response qualitatively similar to that of neonatal mammals (Mortola, 2001). Since CO2 was only de1ivered to the CAM compartment, thus, bypassing intra-airway chemoreceptor stimulation and because the CAM is thought to lack a nerve supply (Windle et al., 1938), the presence ofa \TE response to C02 in our experimental conditions should emphasize that, in the chick embryo, the systemic receptors, either peripherally or centrally located, or both, are functionally active. In conclusion, the externally pipped chick embryo demonstrates a clear ventilatory response to C02, whereas the response to changes in oxygenation is small or absent. Until data from embryos ofother species are collected, it is impossible to exclude that this may be a species-specific characteristic. It is also possible that, in the avian embryos, at least from the view point ofthe regulation ofbreathing, the sudden exposure to atmospheric air with the onset ofexternal pipping may represent an event comparable to that ofbirth in mammals. In mammals, the onset and establishment ofregular breathing at birth bring about a rapid and major increase in arterial P02 (Mortola, 2001). This phenomenon is thought to silence the arterial chemoreceptors and hence, their reflex \TE responses. Within the next few days, however, the receptors gradually reset to the new arterial P02 levels and increase their sensitivity to hypoxia (Blanco et al., 1984, 1988; Kumar and Hanson, 1989; Hertzberg et al., 1990). In the avian embryo, the external pipping phase represents a change from the rebreathing condition ofthe air cell, with low 02 and high C02 levels, to atmospheric air. This transition evokes a rapid 55 increase in arterial oxygenation from the low, pre~pipping values (Tazawa, 1971; Pettit and Whittow, 1982b) to those ofthe young, postnatal chick (Weiss et al., 1965). This acute increase in oxygenation is the basis for the important increase in metabolic rate, which on the one hand helps in establishing \TE, but at the same time may momentarily reduce the sensitivity ofthe chemoreceptors. Ifthis interpretation is correct, from the perspective ofmetabolic and respiratory control, the last phases ofdevelopment in the avian embryo could be considered homologous to the early postnatal phases in mammals. l l Table 4.1 Average values oftotal oxygen consumption CVOztotal, ml kg- min- ) in air and in various gas mixtures o Weight Ta VOz VOz with change P (g) m aIr altered gases (%) Air 10 41.0 (±1) 39.0 (±O.S) 13.1 (±0.6) S%COz 10 39.0 (±0.4) 12.3 (±0.6) 94 (±2) <0.04 Air 10 40.S (±1) 39.4 (±0.2) 11.6 (±0.6) 10% Oz 10 39.3 (±0.2) S.6 (±0.8) 48 (±7) <0.002 Air 10 39.S (±1) 39.3 (±0.3) 13.8 (±0.9) 100% Oz 10 39.1 (±0.2) 13.6 (±0.7) 99 (±3) ns o Values are means ± 1 SEM. Ta, ambient temperature. VOz, oxygen consumption. P, levelof o statistical significance for the comparison ofVOztotal between the air and the experimental gas (two- tailed paired t test); ns, not statistically significant (P>O.OS). Nl, number ofembryos, studied in sets of two. 160 IlS2SZSI Hypercapnia 1 * .--= S 140 x e * x 'o *... Txx ~= X xx Cl 120 x x ~ X xx X ., xxx X ~ ... >< ~ r.r.J. ~ x xx o x X = x x~ X~X xx Q.. )( )C"X ~. r.r.J. 100 x x ~ ~ ~~V )( xx ~ x >< x x x x xxx X xx x x x x xx x 80 xx xx X xx xx r>l' X} 3c " u " x x V02 VC02 VE VEN02 VENC02 (lungs) (lungs) (lungs) (lungs) Figure 4.1. Effects of hypercapnia (5% CO2 in normoxia) exposure to the chorioa1lantoic compartment on the gaseous exchange of the lungs, oxygen consumption CV021ung) and carbon dioxide production (VC02Iung), pulmonary ventilation (VE) and their ratios. Columns indicate mean values, bars represent standard errors. *, significantly different from the values in air (100%, dotted line). _ Hypoxia r;;:::s::sJ Hyperoxia 120 * * .---~ ~ 0 100 ==J- O ~= = 80 ~ --~ 0 c.= ~ ~ 60 ~ 40 V02 VC02 VE VEN02 VENC02 (fungs) (fungs) (fungs) (fungs) Figure 4.2. Effects of hypoxia (10% O2) or hyperoxia (100% 02) of the chorioallantoic compartment on the gaseous exchange of the lungs, oxygen consumption CVo2!ung) and carbon dioxide production CVco2!ung), pulmonary ventilation (\TE) and their ratios. Columns indicate mean values, bars represent standard errors. *, significantly different from the values in air (100%, dotted line). Figure 4.3. Percent changes of tidal volume (at lefl) and breathing frequency (at right) during hypercapnie (5% COz), hypoxic (10% Oz) or hyperoxic (100% Oz) conditions in the CAM compartment only. Columns represent mean values, bars are standard errors. *, significantly different from the values in air (100%, dotted line). Chapter 5 Summary and Conclusions 57 5. Summary and Conclusions 5.1. An Overview ofthe Model During embryonic development, the chick undergoes three distinct phases with respect to gas exchange. Firstly, the prenatal phase; a phase whereby the majority of growth and development takes place. It is characterized simply by the diffusional gas transport between the environment and the chorioallantoic membrane (CAM) across the pores ofthe eggshell. Thus, the CAM is the principal respiratory organ for the majority of incubation in the chick embryo. Secondly, the postnatal phase, where gas exchange is accommodated solely via the convection ofthe lung. This occurs moments prior to hatching. And thirdly, the pipping phase; encompassing both forms ofgas transport. Here a graduaI transition takes place from a purely diffusive respiratory system to the convective respiratory system familiar to all adult birds and mammals. Respiratory muscle contractions have been shown to begin several days before the hatching process is complete, however the lungs do not become inflated until the very last stages ofembryonic development. In the chick embryo, internaI pipping marks the onset of pulmonary ventilation ('VE), although the exact mechanisms that initiate VE still remain unclear. It commences at approximately day 20 ofincubation, at which time the beak pierces the inner shell membrane lining the bottom ofthe air cell. This offers a chance ta improve the gas exchange rate ofthe embryo, which is vital for the efforts required for a successful hatch. However, as long as the eggshell stays intact, the efforts remain confined to a diffusionallimitation and the developmental increase in oxygen consumption (Vo2) results in a progressively hypoxic and hypercapnic condition in the 58 air cell. Thus, after several hours ofrebreathing air cell gas, the embryo enters the external pipping phase and is stimulated to crack the shell and begins breathing directly from the atmosphere (Visschedijk, 1968a,b,c). From the moment the lungs become functional until the time ofhatching, the embryo utilizes two systems ofrespiration. 5.2. The Goals ofthe Study The aims ohhis work were to investigate several critical components responsible for the regulation ofbreathing in the chick embryo during its last stages ofincubation. The fi.rst study looked at the metabolism-ventilation relationship determined by a dual gas exchanging model and how changes in body temperature affected this coupling. The second study revealed the potential mechanical implications on the respiratory system offered by the curled up posture ofthe embryo as weIl as the shell and its accompanying membranes. And thirdly, the presence ofchemosensory activity at this early stage of development was addressed. 5.3. The Metabolic Aspect We asked to what extent, in the chick embryo, do changes in metabolic rate affect pulmonary ventilation during the last two days ofincubation. In mammals, gas exchange can only occur through the lungs, thus a tight metabolism-ventilation coupling may be expected to develop. This relationship, in fact, is well established and can be seen in many conditions ofhypermetabolism as well as hypometabolism. In the chick embryo at term, however, a unique dual gas exchange system exists, hence offering an opportunity for the gaseous metabolism ofthe embryo to be taken care ofby a source other than the 59 lungs. This scenario propelled the question at the core ofthis study. Iftotal metabolic rate, determined by the rates ofall the chemical reactions occurring throughout the embryo, determined the \TE level, then one might expect the changes in total metabolism to be accompanied by proportional changes in \TE. If, on the other hand, the levels of02 and CO2 were the determining factors dictating the changes in \TE, it is plausible that the metabolic coupling to \TE would be more c10sely related to only that component of gaseous metabolism that is not exchanged across the eggshell (i.e. through the CAM). The results ofthis study c1early indicated that, first, increases in total metabolism were accompanied by changes in both tidal volume (VT) and breathing rate (f) and second, and more important, that \TE was more c10sely coupled to that component of gaseous metabolism exchanged through the lungs than to total metabolic rate. This result indicated the efficiency ofthe pulmonary system in that \TE increased when the metabolic requirements ofthe embryo exceeded the capabilities ofthe CAM and proportionately exchanged only that component ofgas not taken on by the membrane. This was valid not only in circumstances ofincreased metabolism due to an increase in egg temperature, but also with the natural progression ofembryonic development. Therefore, the common relationship between pulmonary \TE and gas exchange ofall homeotherms is already verified in the chick embryo, even before it is forced, by the completion ofhatching, to be fully dependent on the lungs. In addition, these findings suggest the likelihood that ventilatory control mechanisms are more intimately connected to peripheral cellular needs via the gaseous component oftissue metabolism as opposed to neural or humoral stimuli originating from the peripheral tissues. 60 5.4. Mechanical Aspect Although the underlying physiology may very weIl be similar, there are obvious differences between birds and mammals with respect to the onset oflung ventilation. For instance, in the newborn mammal, there is an immediate dependence on its own capabilities for gaseous exchange, hence the lungs aeration at birth must occur rapidly. In the avian embryo, on the other hand, the onset oflung ventilation begins not oruy weIl before hatching, but the lungs becomes fully aerated much more graduaIly. As weIl, the embryo relies greatly, especially at the beginning ofthe pipping phase, on the gas exchange provided by the CAM, which continues to function almost until the moment the chick hatches. The lungs in birds are non-expansible and stiffand, in contrast to mammals, where the rib cage and the abdominal cavity are separated into two compartments by the diaphragm, birds do not have a diaphragm. Rather, they possess a single thoracoabdominal cavity that contains multiple air sacs. The coordinated inflation and deflation ofthe interspersed sacs are responsible for pumping air through the lungs. The function ofthe latter is what may be restricted by the curled-up posture ofthe embryo. In this study, we asked towhat extent does the confinement ofthe egg and the curled-up posture hinder the expansion ofthe air sacs. Ifthe respiratory system were mechanically constrained by the posture ofthe embryo, then exteriorizing the embryo from the shell would result in a drop in pressure across the system and in turn, increase the airflow to the lung. Hence, exteriorization would lead to a decrease in impedance (Zrs). In the internaI pippers, freeing the embryo from the eggshell resulted in a significant increase in compliance (Crs) and reduction in expiratory resistance (Rrs). 61 Rence, the embryo's exteriorization did result in changes in the mechanical properties of the respiratory system. Rowever, at the stage ofdevelopment when this occurred (internaI pipping), the CAM is the primary respiratory organ and the lungs contribute very little to total gas exchange. Thus, differences in respiratory mechanics probably have little practical importance on the respiratory efficiency ofthese embryos. The results from exteriorization ofthe external pippers were ofparticular interest. At this stage, the \TE pattern is constant and the lungs are the major contributors to total gas exchange. Ifthe curled posture prevented the proper expansion ofthe air sacs at this stage ofdevelopment, the respiratory mechanical properties would be compromised and hence, affect lung gas exchange. The results indicated that the curled posture did not have any bearing on the mechanical properties ofthe system. We conclude that mechanical factors related to the embryo's posture are unlikely to affect the relationship between metabolism and ventilation during the last days of incubation. 5.5. Chemosensitivity The likelihood that chemosensitivity plays a role in monitoring ventilatory activity at the embryonic level was questioned. Even in marnmalian physiology, the role ofthe blood partial pressure ofcarbon dioxide in regulating and maintaining appropriate levels ofventilation in accordance with metabolic level is an idea cornmonly proposed by physiologists. It is known that adult birds possess chemosensitive reflex responses to both oxygen and carbon dioxide; however, almost nothing was known about the chemical control ofrespiration during the period between pipping and hatching. This study set out 62 to investigate the possibility that VE chemosensitive mechanisms may already be functioning near the end ofincubation and thus may play a role in determining how ventilation is regulated in the chick embryo. The results indicated that, during the external pipping phase ofdevelopment, that is when lung gas exchange is the predominant respiratory organ, there is a clear hyperventilatory response to carbon dioxide, whereas the response to changes in blood oxygenation is limited or absent altogether. An important consideration before concluding that the externally pipped chick embryo lacks chemosensitivity to oxygen is that, prior to breaking the eggsheIl, the embryo is rebreathing from the air ceIl, an environment that has become increasingly hypoxic and hypercapnic since the onset of VE. Thus, exposure to environmental air after rupturing the eggshell could be considered an event comparable to that ofbirth in mammals. In the newborn mammal, birth brings about a large and overwhelming increase in arterial POz. This exposure is said to inhibit the ventilatory reflex response and not until a few days later does the sensitivity ofthe arterial chemoreceptors resume. It is conceivable that the large increase in oxygenation faced by the embryo after external pipping may have the same effect. Therefore, in terms ofrespiratory control, the chemosensitivity ofthe externally pipped embryo may resemble that ofthe newborn mammal. AppendixA Effects of Temperature on Oxygen Consumption Throughout Incubation 64 Appendix A: Effects ofTemperature on Oxygen Consumption Throughoutlncubation Throughout the incubation ofthe egg, there is an exponential increase in oxygen demand due to the increasing metabolic needs required for the growth and development ofthe embryo. At around day 18 ofincubation, oxygen consumption CVÛ2) begins to plateau and changes relatively little until pipping occurs (Burton and Tullett, 1985). The effects oftemperature on metabolism in the chick embryo have been weIl documented (Tazawa, 1973, Nair et al., 1983, Tazawa et al., 1988). Because birds, at this stage ofdevelopment, do not possess the ability to maintain a constant body temperature, cold stress does not stimulate an increase in metabolism for the generation ofheat, as it would be expected for adult birds or mammals. Rather, metabolism slows down, thus leading towards a dec1ine in Vû2. On the other end, heat stimulates an increase in metabolic rate and accordingly, increases the oxygen demands ofthe embryo. To verify the effects oftemperature on metabolism throughout the late stages of development, a similar setup and protocol to that described in Chapter 2 was used. The difference lies in that total embryo VÛ2 was measured, rather than Vû21ung and VÛ2CAM separately. This was achieved by neglecting the parafilm separation at the level ofthe air cell (refer to section 2.3.2.). Sixty embryos were used ranging from days 16 to 21 of incubation (N=1O for each day) and each embryo was exposed to 5 different ambient temperatures set by the water bath (30, 33, 36, 39, 42 OC) starting from the lowest 65 temperature. Temperature equilibration was allotted for between each increment and followed by measurements of\102. Refer to section 2.3.5. for the calculation of\102. The results offigure Al (top panel) indicate that for a given age ofincubation, \102 increases with temperature. Furthermore, between days 20 and 21 ofincubation, the increase in V02 from 30 oC to 42 oC is much larger. This increase in oxygen intake is possible because the embryo is in the external pipping phase, implying that the embryo is breathing directly from the atmosphere, eliminating the diffusional constraints ofthe porous shell. The oxygen limitation provided by the shell toward the end ofincubation is again emphasized in figure Al (bottom panel). Here, \102 is plotted per embryo weight o1 (ml·mino1·kg ) as a function ofincubation age. As the embryo progresses towards hatching, its \102 per embryo weight declines despite the embryo's continuaI development and increasing demand for oxygen required for the hatching process. However, upon external pipping, this decline is halted and the \102 per embryo weight shoots up. This indicates that the progressive drop in the embryo's \102 with incubation is contributed by a diffusionallimitation offered by the shell. 0.6 ----- T30 --0- T33 0.5 --....- T36 ,-...., -v- T39 --- T42 .-e= 0.4 ...... - 0.3 '-"e M 0 0.2 > 0.1 0.0 .-,-....,= 16 e 14 ...... !! 12 ...... -e 10 '-" ~ ~ 8 0 è: 6 J:;. e 4 ~ ...... 2 M 0 0 > 15 16 17 18 19 20 21 22 incubation time (days) Figure A.l. Total oxygen consumption CV02total, top panel, Y-axis) and V02total per embryo weight (bottom panel, Y-axis) plotted against the last days of chick embryo incubation (X-axis) at five different body temperatures ranging from 30 to 42 oc. Each embryo was studied at aIl five temperatures. Every point represents a mean value (N = 10), bars are standard errors. Appendix B The Barometric Method 67 Appendix B: The Barometric Method 1. The Concept Pulmonary ventilation (VE) is the product of tidal volume (VI) and breathing frequency (f). For this project, measurements OfVE were acquired using an adaptation of the barometric method for the calculation of VI while f was collected directly from the chart recordings. The barometric method is a technique that permits measurements ofVI without hindering or interfering with the animal's comfort and weIl being. The model consists of an animal in a sealed chamber (a "box") where the temperature and relative humidity (RH) in the chamber differ from the animal's body temperature and RH. When air from the chamber is inspired, it is warmed and humidified to the pulmonary values, thus causing the gas to expand. As a result, the total pressure in the box increases. The opposite occurs upon expiration (Mortola and FrappeIl, 1998). Rence, the changes in total box pressure in accordance with breathing result in pressure oscillations, which are proportional to the difference in volume from chamber to pulmonary conditions. The volume ofair exposed to the airway conditions (VIa) can be expressed using the laws of ideal gases, VIa = VIc[Ta(PB - PCH2o]/[Tc(PB - PaH20)] Where, VIa = the volume from airway conditions VIC = the volume from chamber conditions Ta = airway temperature 68 PB = barometric pressure PCH20 = partial pressure ofwater in the chamber Tc = chamber temperature PaH20 = partial pressure ofwater in the airways The pressure signal (A) depends on several factors. For example, the smaller the difference between lung and chamber conditions, the smaller the oscillation. Therefore, in order to correctly calculate VT, the A signal needs to be adjusted by a factor (Ga) to account for the gas expansion relative to the airway conditions. This can be seen as: Ga = VTaI (VTa - VTC) Furthermore, the A signal depends on the total volume ofthe chamber. That is, the larger the chamber, the more dampened the signal will be. Thus, the A signal must be multiplied by another factor, K, acknowledging the volume calibration. This can easily be obtained by injecting a known volume (Vcal) into the chamber and record the corresponding change in chamber pressure (PcaI)o Therefore, VT = A· K Ga And by replacing airway values with body values (example, Th for Ta), the complete barometric method equation for calculating VT is as follows: VT = A(Vcal/ PCal)[Th(PB - PCH20)]/{[Th(PB - PCH20] - [Tc(PB - PbH20)]} 2. The Limitations Without careful awareness ofpossible sources oferror, the simplicity ofthis model can easily lead one toward a miscalculation ofVT. For instance, the animal in the 69 chamber must rest quietly in order to minimize muscle activity since any movement may modify the chamber pressure and thus, alters the perception ofthe breathing signal. Secondly, the chamber volume should be small in relation to the animal in order to maximize the signal-to-noise ratio. However, this requirement also needs to be balanced against the comfort ofthe animal and the duration ofthe recording. In a small, sealed chamber, the likelihood for rebreathing to occur is more rapidly reached. As well, thermal drifts due to heat production from the animal are more noticeable in small versus large chambers. A pressure transducer with a high sensitivity and a fast response time is vital, especially when small animaIs with high frequency breathing are used. Tc and Th are the variables that introduce the highest probability for error when using this technique. An inaccurate estimation ofthe temperature is not only consequential since its absolute value is used in the formula, but it also introduces an error in the corresponding water vapor pressure, another variable required in the formula. It is advantageous to have a large temperature gradient between the chamber and pulmonary conditions to minimize the pollution ofthe pressure signal. On the other hand, a small gradient does not imply that the barometric method should not be considered an appropriate technique, it simply entails careful awareness and accuracy. The specifics regarding the application ofthis methodology to the current experiments on chick embryos are given in section 2.3.4. Appendix C Identification of UCP - -- actin uep ... 1 2 3 4 5 6 7 8 Figure C.I. Identification, in the chick embryo, of the actin and un coupling protein (UCP) band by colour precipitation after transfer to membrane and immunoblot (Western blot). Standard Western blot techniques were used. For details on the methodology, refer to Mortola and Naso (1997). Lanes 1 and 8 are brown adipose tissue rat samples, used as markers for the actin and UCP band. Lanes 2-7 are chick embryo samples from, respectively, subcutaneous fat, back muscle, left lung, right lung, liver and pectoral muscle. The presence of actin in aIl tissues is evident, however, it is clear that in the various chick embryo samples tested, there is no indication of UCP. Glossary 72 Glossary air cel! An air pocket formed at the blunt end ofthe egg due to the escape ofwater via the pores ofthe eggshell Air sacs Thinned-wa1led, poody vascularized structures on either side ofthe lungs that serve as bellows to push air unidirectionally through the lungs Carbon dioxide production The volume ofcarbon dioxide produced per unit time Chemoreceptor A receptor that senses chemical stimuli Chemosensitivity The ability to respond to changes in oxygenation and to altered levels ofcarbon dioxide Chorioal!antoic membrane A vascular membrane, formed from the fusion ofthe allantois and chorion; it lines the inner surface ofthe eggshell and provides the only route for gas exchange until the initiation ofpulmonary ventilation Compliance The change in volume per unitary change in pressure; an index ofthe stretchability ofthe organ (i.e. 1 / elastance) Conductance Flow generated by a unitary change in pressure (i.e. 1 / resistance) Convection Movement ofmolecules resulting from the movement of the fluid from which they are part of Diffusion Movement ofmolecules as the result ofa pressure gradient Elastic load An additional elastance confronted during breathing 73 Externalpipping A phase in avian embryo development where the beak cracks the eggshell and begins to breath direcdy from the atmosphere Frequency The number ofbreaths per unit time Homeotherm An animal with the ability to maintain a constant body temperature Hypercapnia An increase in the arterial partial pressure ofcarbon dioxide Hyperoxia An increased level ofoxygenation Hyperpnea An absolute increase in ventilation Hyperventilation An increase in pulmonary ventilation relative to total metabolic rate Hypoventilation An decrease in pulmonary ventilation relative to total metabolic rate Hypoxia A reduced level ofoxygenation Impedance Change in mean total pressure per unitary flow Inertance Acceleration-dependent resistance Inflation time Time taken to inflate the lungs Internai pipping A period during avian embryo development where the beak tears the air cell membrane and initiates pulmonary ventilation Metabolic rate Average speed ofthe metabolic reactions 74 Metabolism AlI the chemical processes occurring within a living orgamsm Oxygen consumption The amount ofoxygen consumed by an organism per unitary time Pneumotachograph A mechanical device used for measuring air flow Poikilotherm An animallacking the ability to maintain a constant body temperature, thus having a direct correlation with the environmental temperature QlOfactor A numerical index ofthe changes ofa variable as a function oftemperature; the change in reaction velocity for a 10 oC change in temperature Resistance The change in pressure required to generate a unitary change in flow Tidal volume The volume ofair inhaled with each breath Time constant, expiratory Determines how rapidly the system will deflate; considers compliant and resistive properties ofthe system Ventilation The volume ofair entering or leaving the lungs per unit time Reference List 76 Reference List Ar, A, Girard, H., Rodeau, I L., 1991. 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