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Oxygenation and establishment of thermogenesis in the avian embryo
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Life Sciences 82 (2008) 50 – 58 www.elsevier.com/locate/lifescie
Oxygenation and establishment of thermogenesis in the avian embryo Kirsten Szdzuy, Laura M. Fong, Jacopo P. Mortola ⁎ Department of Physiology, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada H3G 1Y6 Received 20 April 2007; accepted 18 October 2007
Abstract The production of heat (or thermogenesis) and its response to cold improve very quickly around birth in both mammals and birds. The mechanisms for such rapid perinatal development are not fully understood. Previous experiments with hyperoxia suggested that, during the last phases of incubation, eggshell and membranes might pose a limit to oxygen availability. Hence, it was hypothesized that an improvement in oxygenation by opening the eggshell may contribute to the establishment of thermogenesis. Thermogenesis and its response to cold were measured by indirect calorimetry, in warm (38 °C) conditions and during 1-h exposure to 30 °C. Both improved throughout the various phases of the hatching process. During the latest incubation phases (internal pipping, IP, and star fracture of external pipping, EP), the removal of the eggshell in the region above the air cell raised metabolic rate both in warm and cold conditions (in IP) or the thermogenic response to cold (in EP). Adding hyperoxia after opening the eggshell caused no further increase in the thermogenic response. In cold-incubated embryos thermogenesis during the EP phase was much less than normal; in these embryos, increasing the oxygen availability did not improve thermogenesis. We conclude that oxygenation contributes to the maturation of the thermogenic mechanisms in the perinatal period as long as these mechanisms have initiated their normal developmental process. © 2007 Elsevier Inc. All rights reserved. Keywords: Embryonic development; Oxygen consumption; Perinatal events; Thermoregulation
Introduction In birds and mammals, the transition between fetal and postnatal life causes important changes in many organs and functions, with influences on metabolic, hormonal and cardiorespiratory control. Also thermoregulation is one function undergoing rapid changes. In particular, the ability to produce heat in response to cold, or thermogenic response, improves very quickly after birth in human infants (Hey, 1969) as in other newborn mammals (for review Mortola, 2001) and birds (e.g., Freeman, 1964; Dunn, 1976; Tazawa and Rahn, 1987; Nichelmann and Tzschentke, 2002; Mortola and Labbè, 2005; Dzialowski et al., 2007). The mechanisms behind the rapid development of thermogenesis at birth are not clear. The role of thyroid hormones emerged from the observation that experimental treatment with thiourea blocked the onset of thermogenesis, in bird embryos close to term (Tazawa et al., 1989) and ⁎ Corresponding author. Tel.: +1 514 3984335; fax: +1 514 3987452. E-mail address: [email protected] (J.P. Mortola). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.10.007
in hatchlings (Freeman, 1971). Another contributing factor to the establishment of thermogenesis could be the .rise in oxygenation. In fact, at birth, oxygen consumption (V O2) increases both in mammals (Mortola, 2001) and birds (e.g., Tazawa and Rahn, 1987; Nichelmann and Tzschentke, 2002; Mortola and Labbè, 2005), a phenomenon attributed to the lifting of the prenatal limitation in O2-supply (Tazawa et al., 1988). In the emu embryo, a species with an early development of thermogenic competence, during the internally pipping phase lowering the egg shell temperature to 30 °C did not increase . V O2, but it did when the cold stimulus was accompanied by exposure to 40% O2 (Dzialowski et al., 2007). To what extent the improved oxygenation that normally accompanies the natural process of birth or hatching contributes to the establishment of thermogenesis is not known. Also unknown are the effects of oxygenation on thermogenesis in embryos “small-forgestational-age”, i.e. in growth-retarded embryos close to birth. These questions are difficult to approach experimentally in mammalian preparations, because the transition between placental and pulmonary gas exchange occurs very rapidly. Differently, in
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birds, the transition between chorioallantoic and pulmonary gas exchange is spread over many hours, during which time the embryo begins to pip into the air cell (internal pipping), through the eggshell (external pipping) and eventually completes hatching. Hence, the avian model offers an opportunity to study the development of thermogenesis throughout the various phases, and to investigate how thermogenesis is influenced by an increased oxygenation. Furthermore, cold exposure delays the normal developmental trajectory of thermogenesis (Minne and Decuypere, 1984; Givisiez et al., 2003; Nichelmann, 2004; Tzschentke and Nichelmann, 1999; Tzschentke et al., 2001; Black and Burggren, 2004; Mortola, 2006), providing a model for the effect of oxygenation on thermogenesis in small-for-gestational-age embryos, when the temporal progression of hatching and thermogenesis are mismatched from each other. In this study, first, we precisely defined the development of thermogenesis in the chicken embryo during the phases surrounding hatching. Second, we asked to what extent lifting the limitation imposed by the eggshell on O2 diffusion might contribute to the establishment of the thermogenic mechanisms. To this end, we measured the thermogenic response in embryos during the pipping phases of the hatching process, before and after having artificially opened the eggshell in the region covering the air cell, an intervention that increases the partial pressure of O2 in the allantoic vessels (Nakazawa and Tazawa, 1988). Finally, we performed these measurements in a set of cold-incubated embryos, to test the effects of oxygenation in embryos at term with blunted thermogenic capacity. Materials and methods Experiments were conducted on freshly laid fertilized eggs of White Leghorn chickens (Gallus gallus), obtained from a local supplier. Incubation started around midday (day 0). The eggs were weighed and placed into incubators (Hova-Bator, Savanah, GA, USA), at the temperature (T) of 38 °C and 60% relative humidity, with automatic rotation (90°) four times per day. A T-data logger and a hygrometer were placed inside the incubators. The T-data logger collected the T-value every 10 min, while humidity was read daily. Egg weight at the start of incubation, embryo weight, and the daily water losses (calculated from the drop in egg weight) were measured for each egg. At the end of the experiment, the embryo was killed by exposure to CO2 and cold, the egg was opened, and body weight (W) was measured on a digital scale. Gaseous metabolism Measurements of metabolic rate were obtained by indirect calorimetry, i.e.. by measuring gaseous metabolism .(oxygen consumption, V O2, and carbon dioxide production, V CO2), by the open-flow method (Frappell et al., 1992), adapted to the chicken embryo (Menna and Mortola, 2002, 2003; Mortola and Labbè, 2005). The eggs or the hatchlings were individually placed in a 300-ml plastic container maintained at the desired T by a water bath. A steady gas flow of 100 ml/min for eggs or 200 ml/min for hatchlings was continuously delivered through
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the respirometer, and controlled by a precision flow meter. After passing through a drying column, the inflowing and outflowing gases were monitored by calibrated gas analyzers (O2 anlyzer, Foxbox, Sable Systems Int., Las Vegas or OM-11, Beckman; CO2 analyzer, CD-3A, Applied Electrochemistry) arranged in series. Outflowing gases were monitored continuously, while the inflowing gases were sampled at the onset and at the end of the experiment and at appropriate time intervals, to check for the stability of the analyzers. The output of the analyzers was . displayed on a computer monitor during online acquisition. V O2 . and V CO2 were computed from the flow rate and the inflow– outflow concentration difference, as averages of 10-minute intervals. The values, calculated at standard temperature, pressure and dry conditions, are presented in ml/min (1 ml STPD of O2 = 0.0446 mmol O2, or about 4.8 cal). . Continuous V O2 measurements during the hatching process In a first group of 5 eggs, metabolic rate was measured at the normal incubation T (38 °C) continuously throughout the whole hatching process, starting from the internally pipping (IP) phase, verified by projecting a strong light beam on the egg (egg candling), up to the first 16 h after hatching. The eggs were placed individually in a humidified respirometer, where they stayed for at least one day, during which time the embryo passed through the various phases of hatching. The temperature of the respirometer, maintained around 37.5 °C by a circulating water bath, was monitored by telemetry with a small transmitter placed close to the egg and powered by an external energiserreceiver unit (4000E, Mini Mitter, Sunriver, OR), recorded by standard telemetric techniques. The data of egg temperature, O2 and CO2 concentrations were acquired continuously, displayed online, and analysed at 30-s intervals for about 12 h before and 12 h after hatching. The baseline of the analysers was checked at periodic time intervals to verify the stability of the analysers. Thermogenic response at the various phases of the hatching process The measurements of the thermogenic response were performed at embryonic days E19 to E21 before internal pipping, after internal pipping, during the star fracture of the externalpipping phase, and in the hatchlings during the first 8 h and during the remaining 16 h of the first post-hatching day. These groups will be referred to as pre-IP, IP, EP, H1 b 8 h and H1 N 8 h, respectively. The pre-IP and IP phases were established by candling the egg and verified after opening the eggshell at the end of the experiment. Each animal was studied at only one age. After 20-min acclimatization to the respirometer, gaseous metabolism was measured continuously for 30 min at 38 °C, and for 1 h following an abrupt drop to 30 °C, achieved by an appropriate decrease in the circulating water bath. For comparison purposes, similar measurements were performed also on some embryos at day 11 (E11), i.e. at a time when the embryos are completely ectothermic (Mortola and Labbè, 2005).
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Thermogenic response with increased O2-conductance In IP and EP embryos (N = 10 each), the thermogenic response was measured twice, before and after the eggshell conductance to O2 was increased by removing a portion of the eggshell. The artificial opening was done at the blunted end of the egg, in the region above the air cell, by removing the eggshell over an area of approximately 1 cm2. Then, the egg was returned to 38 °C for 1 h, after which time the cold challenge was repeated. The effect of removing a 1 cm2 eggshell on the egg gasconductance was assessed by measuring water vapour conductance as proposed by Ar et al. (1974). Eggs either intact or with the shell opened, and matched by initial weight, were simultaneously placed in a desiccator kept at constant T (23.5 °C, monitored by a temperature-data logger) and filled by dry KOK pellets, which maintained the humidity close to zero. Because the water pressure gradient between the egg and the surroundings was constant, the water loss (measured by the drop in egg weight over several days) was proportional to the water conductance. Hence, the ratio of the average slope of the daily weight loss between intact and opened eggs represented the ratio of their respective conductances. In an additional set of EP embryos (N = 10), the measurements were performed as described above, except that the runs with the eggshell open (first at 38 °C, then at 30 °C) were performed in hyperoxia (about 35% O2), by delivering the hyperoxic gas inside the respirometer. Control measurements were performed to consider the possibility that the first cold-exposure, with the egg intact, may have some carry-over effects on the second thermogenic response, with the eggshell removed. To this end, measurements of the thermogenic responses were performed twice on control IP and EP eggs (N = 7 per group), following the same protocol of the experimental eggs, but without opening the eggshell. These eggs will be referred to as Controls. Cold-incubation These eggs were incubated at normal T (38 °C) until E18, at which time they were switched to 35 °C and left at this incubation T until they reached the EP phase (star fracture). Then, the thermogenic response was measured before and after the opening and removal of the eggshell, as described above. Quantification of thermogenesis, thermogenic response, and statistical analysis To statistically analyze. the thermogenic responses, first, for each animal, a plot of V O2 versus time was constructed beginning from the last 10 min at 38 °C until the end of the 1h exposure to 30 °C. The graph was printed on fixed coordinates, and the areas under the warm and the cold parts of the curve were digitized with a graphics tablet connected to a minicomputer. The area (ml). was divided by the time duration (min), yielding the average V O2 (ml/min) at 38 °C and at 30 °C; the ratio between the latter and the former gave the average magnitude of the thermogenic response to cold.
. All group data are presented as means ± 1 SEM. Values of V O2 in warm or cold conditions and the thermogenic responses at the various phases were compared among the five groups of animals (pre-IP, IP, EP, H1 b 8 h and H1 N 8 h) by ANOVA, followed by post-hoc Bonferroni's limitations for all possible comparisons. The effect of eggshell opening was evaluated as the ratio between the thermogenic response following and preceding eggshell removal by paired two-tailed t test. The same analysis was done for the comparison between the first and second run of the Control embryos. Finally, the percent changes caused by egg opening (relative to the intact condition) were compared to the percent differences between run 2 and run 1 of Controls by unpaired two-tailed t test. In all cases, significant differences were considered statistically significant at P b 0.05. Results Details about the number and body weight of the animals used for each particular experiment and age group are given in Table 1. Gaseous metabolism at 38 °C Fig. 1 provides . a visual . impression of the changes in gaseous metabolism (V O2 and V CO2) during the hatching process, from the IP phase until 12 .h after hatching. This representative recording shows that V O2 increased throughout the hatching period, with two major abrupt jumps, at the onset of the EP
Table 1 Number of animals, egg weight, age and body weight Age, days
N (N with yolk Egg weight Postincorporated) at E0, g hatching hours
1 — Development of the thermogenic response Pre-IP 19.7 ± 0.3 7 (0) 58.5 ± 2.4 IP 19.9 ± 0.2 17 (8) 58.1 ± 1.4 EP 19.9 ± 0.1 17 (16) 56.0 ± 1.1 H1 b 8 h 20.7 ± 0.3 a 7 (7) 57.1 ± 1.8 H1 N 8 h 20.0 ± 0.0 a 7 (7) 56.1 ± 0.8 E11 b 11 5 (0) 57.7 ± 1.2
– – – 3.1 ± 0.6 20.5 ± 1.1 –
2 — Thermogenic response before and after eggshell removal IP opening 20.3 ± 0.3 10 (6) 56.9 ± 2.0 – IP control 19.4 ± 0.2 7 (2) 59.7 ± 1.6 – EP opening 20.0 ± 0.0 10 (9) 56.3 ± 1.8 – EP control 20.0 ± 0.0 7 (7) 56.4 ± 1.0 –
Body weight, g
30.2 ± 2.2 36.0 ± 1.6 42.0 ± 0.8 40.6 ± 1.4 37.1 ± 0.8 4.0 ± 0.2
37.4 ± 1.9 35.6 ± 1.8 42.5 ± 1.2 40.5 ± 1.5
2 — Thermogenic response before and after eggshell removal, with hyperoxia EP 20.3 ± 0.2 10 (10) 61.3 ± 1.3 – 46.9 ± 0.9 3 — Thermogenic response before and after eggshell removal in cold-incubated embryos EP 20.4 ± 0.2 5 (5) 54.4 ± 2.6 – 40.2 ± 1.9 Values are mean ±1 SEM. N, number of animals. a In the hatchlings, age corresponds to the day of hatching. Pre-IP, IP, respectively before or after internal pipping. EP, during the star fracture of the external-pipping phase. H1 b 8 h, H1 b 8 h, hatchlings respectively during the first 8 h or the remaining 16 h of the first post-hatching day. b At E11, N refers to the number of sets, each set comprising two embryos.
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. . Fig. 1. Continuous measurements of oxygen consumption (VO2) and carbon dioxide production (VCO2) in one representative embryo from 12 h before to 12 h after hatching. The embryo was at the incubation temperature of 38 °C. EP indicates the onset of external pipping, with star fracture of the shell. Interruptions in the continuous lines indicate that the sampling was temporarily halted to check for baseline.
phase (star fracture) and at hatching itself. At this latter time, . V O2 approximately doubled, and gradually declined during the . following hours. The gradual increase in V O2 throughout the hatching phase was also apparent from the mean . values of the 5 groups measured at 38 °C. In fact, resting V O2 averaged about 0.3–0.35 ml/min in the pre-IP and IP embryos, 0.42 ml/min in the EP, and about 0.85 ml/min in the hatchlings (Fig. 2, left panel). The animal body weight (W) was similar among the preIP and IP embryonic stages; then, it increased in the EP embryos and hatchlings, because of the incorporation of the remaining yolk (Table 1).
Thermogenic responses at the various phases of the hatching process At all embryonic phases, exposure .to cold caused a very modest and short-lasting increase in V O2, followed by some decline (Fig. . 2, right panel). Hence, in the pre-IP, IP and EP embryos, V O2during the 1 h of cold averaged less than at 38 °C. Differently, in the hatchlings the thermogenic responses to cold were substantial, and the older hatchlings were able to maintain . V O2 better than the younger hatchlings. For comparison purposes this figure includes also the data of embryos at E11,
. Fig. 2. Left panel: oxygen consumption (VO2) measured continuously during the last 30 min at 38 °C, and one hour at . 30 °C, in embryos at different times of the hatching process, and in hatchlings. The vertical dashed line indicates the onset of the 30 °C exposure. Right panel: the VO2 data in the cold are plotted as percent of the 38 °C value preceding the onset of the cold (100%, horizontal dashed line). The fainter symbols refer to embryos studied at day 11 (E11), at which time they are strictly ectothermic, and the continuous line is the linear regression through the E11 data points. Symbols are mean values of 7 to 17 animals (or 5 sets of 2 embryos each in the case of E11). Bars indicate 1 SEM. Curves with different letters are statistically different from each other.
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passing of days. The average weight loss was 0.25 ± 0.01 g/day in the intact eggs, and 0.99 ± 0.04 g/day in those with eggshell opened (Fig. 3). Hence, opening the eggshell at its blunted end and removing about 1 cm2 increased gas-conductance by approximately 0.99/0.25 = 4 times. Thermogenesis after artificial opening the eggshell
Fig. 3. Weight of eggs maintained at a constant temperature in a desiccator for various days. Open symbols refer to intact eggs, filled symbols refer to eggs with the eggshell removed at its blunted end, for an area of about 1 cm2. The heavy and dashed lines are the linear regressions through the mean values of the intact and opened eggs, respectively.
i.e., at a developmental stage of pure ectothermy. All the curves were displayed above the E11 curve, indicating that also the preIP embryos presented some degree of endothermy, even though their thermogenic response was very modest. Opening the eggshell In the desiccator, at the constant water vapour pressure gradient of 21.3 mm Hg, eggs lost weight linearly with the
. At the IP stage, artificial opening of the eggshell raised V O2, especially during the cold-exposure (Fig. 4, top panel at right). On the other hand, simply repeating the cold exposure without opening the . shell (run 2 versus run 1 in Controls) did not increase V O2; rather, it had a small trend to lower it (Fig. 4, top panel at left). Therefore, the ratios of the thermogenic response with eggshell opened, relative to intact, were significantly higher than the ratios between run 2 and run 1 of Controls (P b 0.05). After normalisation by the values at 38 °C, the shape of the thermogenic curves appeared quite similar, both in Controls and in the experimental embryos (Fig. 4, bottom panels). In conclusion, in the embryos during the IP phase, opening the shell increased . the thermogenic capacity by raising the absolute values of V O2. Differently from the IP, at. the EP stage artificial opening of the eggshell did not alter V O2 at 38 °C, while it caused an important improvement of the thermogenic response (Fig. 5, top panel, at right) (P b 0.001). In Controls (Fig. 5, top panel at left) the differences between run 1 and 2 were not statistically significant. Therefore, in the cold, the ratios of the thermogenic responses with eggshell opened and eggshell intact were
. Fig. 4. Oxygen consumption (VO2) in warm conditions (38 °C) and during the 60 min of cold exposure (30 °C) in embryos during the internal pipping (IP) phase. At left are Controls (N = 7), run 1 and run 2 referring to the two consecutive thermogenic responses. At right are experimental embryos (N = 10), in the intact situation (open symbols), and with the eggshell opened (filled symbols). The bottom panels represent the thermogenic responses, where the values in the cold are expressed as percent of the warm values. The continuous oblique line represents the response of the purely ectothermic embryos, at day E11 (from Fig. 2). Symbols are group means, bars indicate 1 SEM.
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. Fig. 5. Oxygen consumption (VO2) in warm conditions (38 °C) and during the 60 min of cold exposure (30 °C) in embryos during the star fracture of the externalpipping (EP) phase. At left are Controls (N = 7), run 1 and run 2 referring to the two consecutive thermogenic responses. At right are represented the embryos of the experimental group, in the intact situation (open symbols), and with the eggshell removed from the blunted end of the egg (filled symbols). The bottom panels represent the thermogenic responses, where the values in the cold are expressed as percent of the warm values. The continuous oblique line represents the response of the purely ectothermic embryos, at day E11 (from Fig. 2). Symbols are group means, bars indicate 1 SEM.
significantly higher than the corresponding ratios between run 2 and run 1 of Controls (P b 0.05). After normalisation by the warm values, in Controls, the thermogenic response of run 2 differed significantly from that of run 1 (P b 0.01; Fig. 5, bottom panels). These differences were significantly smaller than those observed by opening the eggshell (top left and top . right panels; P b 0.05). In conclusions, not only the overall V O2, but also the thermogenic responses were significantly higher with the eggshell removed than in the intact condition. Eggshell opening and hyperoxia In EP embryos, artificial opening of the eggshell with ac. companying hyperoxia raised V O2 significantly both at 38 °C (P b 0.05) and during the cold exposure (P b 0.01; Fig. 6, top panel). Also the thermogenic response exceeded that measured in the intact eggs (P b 0.05; bottom panel). However, none of these changes differed significantly from those measured in the EP embryos by solely opening the eggshell. Eggshell opening in cold-incubated embryos The embryos that were maintained at 35 °C during the last three days of incubation went through the normal phases of hatching, although with a slight delay compared to those incubated at the normal 38 °C. Their thermogenic response to cold was tested at the time of the EP-star fracture, and it was very modest. In fact, during the last 10–15 min of the 1-h cold
. Fig. 6. Oxygen consumption (VO2) in warm conditions (38 °C) and during the 60 min of cold exposure (30 °C) in embryos during the star fracture of the external-pipping (EP) phase. Open symbols refer to the intact situation, filled symbols refer to the test with the eggshell removed at the blunted end of the egg and additional O2 (about 35% O2). Values are expressed in ml/min (top panel) and in percent of the values attained at 38 °C (bottom panel). Symbols are group means (N = 10), bars indicate 1 SEM.
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embryo being provided by the pregnant mother or by the incubating parent, while it becomes an essential means of thermoregulation after birth. Differently from the cardiorespiratory adjustments, little is known about the factors involved in the rapid development of thermogenesis during the perinatal period. In mammals, catecholamines and thyroid hormones (triiodothyronine) are involved in the maturation of thermogenesis (Power et al., 2004). In birds, the dependency on the thyroid function is supported by experiments showing that treatment with thiourea blocked the onset of thermogenesis (Tazawa et al., 1989; Freeman, 1971). With the current experiment we wanted to consider the role that oxygenation per se might have in the establishment of thermogenesis. The avian model offered the opportunity of exploring the role of oxygenation at precisely defined stages, because of its gradual transition between diffusive and convective gas exchange, and because it is free from the possibility that maternal or placental responses may interfere with the fetal development of thermoregulation. Methodological considerations
Fig. 7. Thermogenic response in embryos incubated at 35 °C during the last three days of incubation. All embryos were studied during the star fracture of the . external-pipping (EP) phase. At top, oxygen consumption (VO2) is expressed in absolute values (ml/min) during warm conditions (38 °C) and during the 1-hour . cold exposure (30 °C). At bottom, values of VO2 are expressed in percent of those at 38 °C. Open and filled symbols, refer, respectively, to the condition of intact eggshell and of eggshell removed at the blunted end of the egg. The continuous oblique line represents the response of the purely ectothermic embryos, at day E11 (from Fig. 2). Symbols indicate group means, bars indicate 1 SEM.
. exposure the percent change in V O2 of these embryos was similar to that of the ectothermic E11 embryos (Fig. 7, open symbols). Opening the eggshell produced no significant change in either thermogenesis or thermogenic response (Fig. 7, filled symbols). . For most of the experiments described above the changes in V CO2 with T, and the differences among groups or. conditions were qualitatively similar to those described for V O2. The EP embryos in hyperoxia represented the exception. In these em. bryos the increase in V in the cold was 21% less than the CO 2 . increase in V O2. Discussion In birds, as in mammals, the transition from prenatal to postnatal life involves abandoning the diffusive form of gas exchange (chorioallantoic or placental) to initiate gas convection (pulmonary ventilation). The success of this transition depends on specific respiratory and cardiovascular adjustments, including the closure of systemic shunts in favour of pulmonary circulation, a process that is under hormonal control but also depends on adequate oxygenation. Thermoregulation is another function drastically changed at birth. In fact, prenatally, endothermy is minimal, the heat requirements of the developing
The important rise in thermogenic capacity between the last phase of embryonic development and the first days after hatching had been reported frequently (Freeman, 1964; Dunn, 1976; Tazawa and Rahn, 1987; Nichelmann and Tzschentke, 2002; Mortola and Labbè, 2005; Dzialowski et al., 2007). However, to assess the role of oxygenation, first, we needed an accurate time-course of the development of thermogenesis and its response to cold throughout the hatching phases. We found, as expected, that also the pre-IP embryos have some degree of endothermy, as shown by a thermogenic level exceeding that of the ectothermic E11 embryos both in warm and cold conditions. However, the thermogenic response improved drastically in the transition from EP to early hatching (Fig. 2). Therefore we have chosen the IP and EP (star fracture) stages as the appropriate phases to examine the effect of oxygenation. Opening and removing the eggshell above the airspace, an intervention that caused some four-fold increase in O2conductance, was the method chosen to increase the embryo's oxygenation. We felt that this was the most appropriate way of mimicking the natural process of increased O2 availability at hatching. The alternate approach would have been to expose the embryos to hyperoxia, as recently done on emu embryos (Dzialowski et al., 2007). However, exposure of the whole chorioallantoic membrane to hyperoxia not only differs from the natural event but also risks diminishing its perfusion, which varies inversely with oxygenation (Strick et al., 1991; Burton and Palmer, 1992), hindering its gas exchange properties. In addition, the egg stores for O2, although probably much smaller than those for CO2 (Mortola and Besterman, 2007), can trap O2 during hyperoxia, giving the false impression of a high . embryonic V O2and low Respiratory Exchange Ratio, a situation . that jeopardizes the calculation of V O2 (Frappell et al., 1991). A potential problem was the fact that the IP and, especially, the EP phases last only a few hours. (see Fig. 1), and are characterized by a progressive rise in V O2 (Fig. 2). Indeed, the
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few-hour experimental periods needed to measure the effects of eggshell opening after the test with the eggshell intact must have included the natural progression of thermogenesis. For this reason we have compared the results of eggshell opening to those of control experiments on intact eggs, subjected to two consecutive runs that temporally matched those of the experimental protocol. Increased O2 conductance A 1-cm2 opening of the air cell may seem a trivial fraction of the total egg surface area of approximately 70 cm2. In reality, it represents a large increase (approximately 4-fold, Fig. 3) in eggshell conductance. Furthermore, opening the eggshell means that the O2 partial pressure (PO2) in the air cell rises by about 50%, from the end-incubation value of about 100 mm Hg (Wangensteen and Rahn, 1971) to 153 mm Hg (at 60% water vapour saturation). Høiby et al. (1983) commented that, even though only about one third of the total embryo's blood flow is exposed to the air cell, its complete O2-saturation would still represent a significant increase (about 16%) to the total O2 content of the arterialised blood returning to the embryo. This agrees with the PO2 values measured in the allantoic vessels of embryos developing in eggs with air cell opened (Nakazawa and Tazawa, 1988). The increase in oxygenation during the IP phase raised . resting V O2 both in warm and cold conditions, without changing their relative proportion. The result conforms to the idea . that, in the chicken embryo at term, O2 availability limits V O2 (Høiby et al., 1983; Burton and Tullett, 1985; Tazawa et al., 1992, also for review). The same has been noted in emu embryos during the . IP phase (Dzialowski et al., 2007). Indeed, the increase in V O2 that we observed by opening the airspace of the IP embryos averaged 12%, very close to the 13% reported by Høiby et al. (1983) with exposure to pure O2. Hence, during the IP phase, the increased metabolism at 38 °C and the increased thermogenesis in the cold after opening the shell can be explained by removal of the O2-limitation. Differently, in the EP embryos . at 38 °C, opening the eggshell did not cause any increase in V O2. At this time of the hatching process the star fracture probably resolved the O2 limitation of the embryo. at rest, as suggested also by the fact that the embryo's V O2 was higher than during the IP phase. On the other hand, the thermogenic response to cold drastically increased after removing the shell, indicating that the diffusion of O2 through the star fracture alone, although providing adequate oxygenation at 38 °C, was still representing a limitation in the cold, when O2 demands. are increased. The exposure to hyperoxia did not raise V O2 significantly more than already obtained by removing the eggshell. Furthermore, the magnitude of the increase in hyperoxia could be. overestimated, owing to the fact that some of the measured V O2 includes O2 physically dissolved in the yolk and other embryonic and extra-embryonic . fluids. Hence, in hyperoxia, changes in V CO2 are more representative of the changes in embryonic . metabolism, and they were smaller than the changes in V O2. In any case, our results showed that, by adding O2, there was no further
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improvement of the thermogenic response compared to the opening experiment without hyperoxia. This indicates that the opening of the eggshell as it occurs during the natural hatching process entirely removes the oxygen limitation, and is fully sufficient for the establishment of thermogenesis. In conclusion, by increasing the improving the embryo's O2 availability . eggshell O2 conductance raised V O2, relieving the O2 limitation that, in the EP phase, precluded the full manifestation of the thermogenic response. Opening the eggshell in cold-incubated embryos Cold-incubation during the last phases of incubation slows down growth and blunts the development of thermogenesis (Mortola, 2006). This was the case also of the cold-incubated EP embryos of this study. Hence, these growth-retarded embryos provided an opportunity to investigate the effect of oxygenation during the EP phase in embryos small for age, and with a more primitive thermogenic capacity. The results indicated that the oxygenation offered by eggshell opening did not improve their thermogenic response, contrary to what observed in normally incubated embryos (compare Figs. 5 and 7). Therefore, the increase in thermogenesis observed in normally incubated embryos by raising their O2 conductance is not a phenomenon time-linked to the hatching process; rather, it is linked to the level of maturity of thermogenesis. In other words, the stimulatory effects of oxygenation on thermogenesis do not depend on hatching and its associated events but on the developmental staging of thermogenesis itself. Therefore, if these data were extrapolated to the human case, one would anticipate that, during birth, oxygenation can improve heat production in infants at term, i.e., in those infants with thermoregulatory mechanisms in the process of maturation, but would be ineffectual on those premature infants who lack thermogenic abilities. Acknowledgement Funds for this research came from a Canadian Institute of Health Research grant. References Ar, A., Paganelli, C.V., Reeves, R.B., Greene, D.G., Rahn, H., 1974. The avian egg: water vapour conductance, shell thickness, and functional pore area. The Condor 76, 153–158. Black, J.L., Burggren, W.W., 2004. Acclimation to hypothermic incubation in developing chicken embryos (Gallus domesticus). I. Developmental effects and chronic and acute metabolic adjustments. Journal of Experimental Biology 207, 1543–1552. Burton, G.J., Palmer, M.E., 1992. Development of the chick chorioallantoic capillary plexus under normoxic and normobaric hypoxic and hyperoxic conditions: a morphometric study. Journal of Experimental Biology 262, 291–298. Burton, F.G., Tullett, S.G., 1985. Respiration of avian embryos. Comparative Biochemistry and Physiology A 82, 735–744. Dunn, E.H., 1976. Development of endothermy and existence energy expenditure of nestling double-crested cormorants. The Condor 78, 350–356. Dzialowski, E.M., Burggren, W.W., Komoro, T., Tazawa, H., 2007. Development of endothermic metabolic response in embryos and hatchlings of the
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emu (Dromaius novaehollandiae). Respiratory Physiology and Neurobiology 155, 286–292. Frappell, P., Dotta, A., Mortola, J.P., 1991. Metabolism during normoxia, hyperoxia, and recovery in newborn rats. Canadian Journal of Physiology and Pharmacology 70, 408–411. Frappell, P., Lanthier, C., Baudinette, R.V., Mortola, J.P., 1992. Metabolism and ventilation in acute hypoxia: a comparative analysis in small mammalian species. American Journal of Physiology 262, R1040–R1046. Freeman, B.M., 1964. The emergence of the homeothermic metabolic response in the fowl (Gallus domesticus). Comparative Biochemistry and Physiology 13, 413–422. Freeman, B.M., 1971. Impaired thermoregulation in the thiouracil-treated neonate fowl. Comparative Biochemistry and Physiology A 40, 553–555. Givisiez, P.E.N., Furlan, R.L., Malheiros, E.B., Macari, M., 2003. Incubation and rearing temperature effects on Hsp70 levels and heat stress response in broilers. Canadian Journal of Animal Science 83, 213–220. Hey, E.N., 1969. The relation between environmental temperature and oxygen consumption in the new-born baby. Journal of Physiology (London) 200, 589–603. Høiby, M., Aulie, A., Reite, O.B., 1983. Oxygen uptake in fowl eggs incubated in air and pure oxygen. Comparative Biochemistry and Physiology A 74, 315–318. Menna, T.M., Mortola, J.P., 2002. Metabolic control of pulmonary ventilation in the developing chick embryo. Respiratory Physiology and Neurobiology 130, 43–55. Menna, T.M., Mortola, J.P., 2003. Ventilatory chemosensitivity in the chick embryo. Respiratory Physiology and Neurobiology 137, 69–79. Minne, B., Decuypere, E., 1984. Effects of late prenatal temperatures on some thermoregulatory aspects in young chickens. Archiv Für Experimentelle Veterinärmedizin 38, 374–383. Mortola, J.P., 2001. Respiratory physiology of newborn mammals. A Comparative Perspective. The Johns Hopkins University Press, Baltimore. Mortola, J.P., 2006. Metabolic response to cooling temperatures in chicken embryos and hatchlings after cold incubation. Comparative Biochemistry and Physiology A 145, 441–448. Mortola, J.P., Besterman, A.D., 2007. Gaseous metabolism of the chicken embryo and hatchling during post-hypoxic recovery. Respiratory Physiology and Neurobiology 156, 212–219.
Mortola, J.P., Labbè, K., 2005. Oxygen consumption of the chicken embryo: interaction between temperature and oxygenation. Respiratory Physiology and Neurobiology 146, 97–106. Nakazawa, S., Tazawa, H., 1988. Blood gases and hematological variables of chick embryos with widely altered shell conductance. Comparative Biochemistry and Physiology A 89, 271–277. Nichelmann, M., 2004. Perinatal epigenetic temperature adaptation in avian species: comparison of turkey and Muscovy duck. Journal of Thermal Biology 29, 613–619. Nichelmann, M., Tzschentke, B., 2002. Ontogeny of thermoregulation in precocial birds. Comparative Biochemistry and Physiology A 131, 751–763. Power, G.G., Blood, A.B., Hunter, C.J., 2004. Perinatal thermal physiology, In: Polin, R.A., Fox, W.W., Abman, S.H. (Eds.), Fetal and Neonatal Physiology, 3rd Edition. Saunders, Philadelphia, pp. 541–548. Strick, D.M., Waycaster, R.L., Montani, J.-P., William, J.G., Adair, T.H., 1991. Morphometric measurements of chorioallantoic membrane vascularity: effects of hypoxia and hyperoxia. American Journal of Physiology 260, H1385–H1389. Tazawa, H., Hashimoto, Y., Nakazawa, S., Whittow, G.C., 1992. Metabolic responses of chicken embryos and hatchlings to altered O2 environments. Respiratory Physiology 88, 37–50. Tazawa, H., Rahn, H., 1987. Temperature and metabolism of chick embryos and hatchlings after prolonged cooling. Journal of Experimental Zoology (Supplement 1), 105–109. Tazawa, H., Wakayama, H., Turner, J.S., Paganelli, C.V., 1988. Metabolic compensation for gradual cooling in developing chick embryos. Comparative Biochemistry and Physiology A 89, 125–129. Tazawa, H., Whittow, G.C., Turner, J.S., Paganelli, C.V., 1989. Metabolic responses to gradual cooling in chicken eggs treated with thiourea and oxygen. Comparative Biochemistry and Physiology A 92, 619–622. Tzschentke, B., Basta, D., Nichelmann, M., 2001. Epigenetic temperature adaptation in birds: peculiarities and similarities in comparison to acclimation. News of Biomedical Science 1, 26–31. Tzschentke, B., Nichelmann, M., 1999. Development of avian thermoregulatory system during the early postnatal period: development of thermoregulatory set-point. Ornis Fennica 76, 189–198. Wangensteen, D., Rahn, H., 1970/71. Respiratory gas exchange by the avian embryo. Respiratory Physiology 11, 31–45.
Life Sciences 82 (2008) 50 – 58 www.elsevier.com/locate/lifescie
Oxygenation and establishment of thermogenesis in the avian embryo Kirsten Szdzuy, Laura M. Fong, Jacopo P. Mortola ⁎ Department of Physiology, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada H3G 1Y6 Received 20 April 2007; accepted 18 October 2007
Abstract The production of heat (or thermogenesis) and its response to cold improve very quickly around birth in both mammals and birds. The mechanisms for such rapid perinatal development are not fully understood. Previous experiments with hyperoxia suggested that, during the last phases of incubation, eggshell and membranes might pose a limit to oxygen availability. Hence, it was hypothesized that an improvement in oxygenation by opening the eggshell may contribute to the establishment of thermogenesis. Thermogenesis and its response to cold were measured by indirect calorimetry, in warm (38 °C) conditions and during 1-h exposure to 30 °C. Both improved throughout the various phases of the hatching process. During the latest incubation phases (internal pipping, IP, and star fracture of external pipping, EP), the removal of the eggshell in the region above the air cell raised metabolic rate both in warm and cold conditions (in IP) or the thermogenic response to cold (in EP). Adding hyperoxia after opening the eggshell caused no further increase in the thermogenic response. In cold-incubated embryos thermogenesis during the EP phase was much less than normal; in these embryos, increasing the oxygen availability did not improve thermogenesis. We conclude that oxygenation contributes to the maturation of the thermogenic mechanisms in the perinatal period as long as these mechanisms have initiated their normal developmental process. © 2007 Elsevier Inc. All rights reserved. Keywords: Embryonic development; Oxygen consumption; Perinatal events; Thermoregulation
Introduction In birds and mammals, the transition between fetal and postnatal life causes important changes in many organs and functions, with influences on metabolic, hormonal and cardiorespiratory control. Also thermoregulation is one function undergoing rapid changes. In particular, the ability to produce heat in response to cold, or thermogenic response, improves very quickly after birth in human infants (Hey, 1969) as in other newborn mammals (for review Mortola, 2001) and birds (e.g., Freeman, 1964; Dunn, 1976; Tazawa and Rahn, 1987; Nichelmann and Tzschentke, 2002; Mortola and Labbè, 2005; Dzialowski et al., 2007). The mechanisms behind the rapid development of thermogenesis at birth are not clear. The role of thyroid hormones emerged from the observation that experimental treatment with thiourea blocked the onset of thermogenesis, in bird embryos close to term (Tazawa et al., 1989) and ⁎ Corresponding author. Tel.: +1 514 3984335; fax: +1 514 3987452. E-mail address: [email protected] (J.P. Mortola). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.10.007
in hatchlings (Freeman, 1971). Another contributing factor to the establishment of thermogenesis could be the .rise in oxygenation. In fact, at birth, oxygen consumption (V O2) increases both in mammals (Mortola, 2001) and birds (e.g., Tazawa and Rahn, 1987; Nichelmann and Tzschentke, 2002; Mortola and Labbè, 2005), a phenomenon attributed to the lifting of the prenatal limitation in O2-supply (Tazawa et al., 1988). In the emu embryo, a species with an early development of thermogenic competence, during the internally pipping phase lowering the egg shell temperature to 30 °C did not increase . V O2, but it did when the cold stimulus was accompanied by exposure to 40% O2 (Dzialowski et al., 2007). To what extent the improved oxygenation that normally accompanies the natural process of birth or hatching contributes to the establishment of thermogenesis is not known. Also unknown are the effects of oxygenation on thermogenesis in embryos “small-forgestational-age”, i.e. in growth-retarded embryos close to birth. These questions are difficult to approach experimentally in mammalian preparations, because the transition between placental and pulmonary gas exchange occurs very rapidly. Differently, in
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birds, the transition between chorioallantoic and pulmonary gas exchange is spread over many hours, during which time the embryo begins to pip into the air cell (internal pipping), through the eggshell (external pipping) and eventually completes hatching. Hence, the avian model offers an opportunity to study the development of thermogenesis throughout the various phases, and to investigate how thermogenesis is influenced by an increased oxygenation. Furthermore, cold exposure delays the normal developmental trajectory of thermogenesis (Minne and Decuypere, 1984; Givisiez et al., 2003; Nichelmann, 2004; Tzschentke and Nichelmann, 1999; Tzschentke et al., 2001; Black and Burggren, 2004; Mortola, 2006), providing a model for the effect of oxygenation on thermogenesis in small-for-gestational-age embryos, when the temporal progression of hatching and thermogenesis are mismatched from each other. In this study, first, we precisely defined the development of thermogenesis in the chicken embryo during the phases surrounding hatching. Second, we asked to what extent lifting the limitation imposed by the eggshell on O2 diffusion might contribute to the establishment of the thermogenic mechanisms. To this end, we measured the thermogenic response in embryos during the pipping phases of the hatching process, before and after having artificially opened the eggshell in the region covering the air cell, an intervention that increases the partial pressure of O2 in the allantoic vessels (Nakazawa and Tazawa, 1988). Finally, we performed these measurements in a set of cold-incubated embryos, to test the effects of oxygenation in embryos at term with blunted thermogenic capacity. Materials and methods Experiments were conducted on freshly laid fertilized eggs of White Leghorn chickens (Gallus gallus), obtained from a local supplier. Incubation started around midday (day 0). The eggs were weighed and placed into incubators (Hova-Bator, Savanah, GA, USA), at the temperature (T) of 38 °C and 60% relative humidity, with automatic rotation (90°) four times per day. A T-data logger and a hygrometer were placed inside the incubators. The T-data logger collected the T-value every 10 min, while humidity was read daily. Egg weight at the start of incubation, embryo weight, and the daily water losses (calculated from the drop in egg weight) were measured for each egg. At the end of the experiment, the embryo was killed by exposure to CO2 and cold, the egg was opened, and body weight (W) was measured on a digital scale. Gaseous metabolism Measurements of metabolic rate were obtained by indirect calorimetry, i.e.. by measuring gaseous metabolism .(oxygen consumption, V O2, and carbon dioxide production, V CO2), by the open-flow method (Frappell et al., 1992), adapted to the chicken embryo (Menna and Mortola, 2002, 2003; Mortola and Labbè, 2005). The eggs or the hatchlings were individually placed in a 300-ml plastic container maintained at the desired T by a water bath. A steady gas flow of 100 ml/min for eggs or 200 ml/min for hatchlings was continuously delivered through
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the respirometer, and controlled by a precision flow meter. After passing through a drying column, the inflowing and outflowing gases were monitored by calibrated gas analyzers (O2 anlyzer, Foxbox, Sable Systems Int., Las Vegas or OM-11, Beckman; CO2 analyzer, CD-3A, Applied Electrochemistry) arranged in series. Outflowing gases were monitored continuously, while the inflowing gases were sampled at the onset and at the end of the experiment and at appropriate time intervals, to check for the stability of the analyzers. The output of the analyzers was . displayed on a computer monitor during online acquisition. V O2 . and V CO2 were computed from the flow rate and the inflow– outflow concentration difference, as averages of 10-minute intervals. The values, calculated at standard temperature, pressure and dry conditions, are presented in ml/min (1 ml STPD of O2 = 0.0446 mmol O2, or about 4.8 cal). . Continuous V O2 measurements during the hatching process In a first group of 5 eggs, metabolic rate was measured at the normal incubation T (38 °C) continuously throughout the whole hatching process, starting from the internally pipping (IP) phase, verified by projecting a strong light beam on the egg (egg candling), up to the first 16 h after hatching. The eggs were placed individually in a humidified respirometer, where they stayed for at least one day, during which time the embryo passed through the various phases of hatching. The temperature of the respirometer, maintained around 37.5 °C by a circulating water bath, was monitored by telemetry with a small transmitter placed close to the egg and powered by an external energiserreceiver unit (4000E, Mini Mitter, Sunriver, OR), recorded by standard telemetric techniques. The data of egg temperature, O2 and CO2 concentrations were acquired continuously, displayed online, and analysed at 30-s intervals for about 12 h before and 12 h after hatching. The baseline of the analysers was checked at periodic time intervals to verify the stability of the analysers. Thermogenic response at the various phases of the hatching process The measurements of the thermogenic response were performed at embryonic days E19 to E21 before internal pipping, after internal pipping, during the star fracture of the externalpipping phase, and in the hatchlings during the first 8 h and during the remaining 16 h of the first post-hatching day. These groups will be referred to as pre-IP, IP, EP, H1 b 8 h and H1 N 8 h, respectively. The pre-IP and IP phases were established by candling the egg and verified after opening the eggshell at the end of the experiment. Each animal was studied at only one age. After 20-min acclimatization to the respirometer, gaseous metabolism was measured continuously for 30 min at 38 °C, and for 1 h following an abrupt drop to 30 °C, achieved by an appropriate decrease in the circulating water bath. For comparison purposes, similar measurements were performed also on some embryos at day 11 (E11), i.e. at a time when the embryos are completely ectothermic (Mortola and Labbè, 2005).
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Thermogenic response with increased O2-conductance In IP and EP embryos (N = 10 each), the thermogenic response was measured twice, before and after the eggshell conductance to O2 was increased by removing a portion of the eggshell. The artificial opening was done at the blunted end of the egg, in the region above the air cell, by removing the eggshell over an area of approximately 1 cm2. Then, the egg was returned to 38 °C for 1 h, after which time the cold challenge was repeated. The effect of removing a 1 cm2 eggshell on the egg gasconductance was assessed by measuring water vapour conductance as proposed by Ar et al. (1974). Eggs either intact or with the shell opened, and matched by initial weight, were simultaneously placed in a desiccator kept at constant T (23.5 °C, monitored by a temperature-data logger) and filled by dry KOK pellets, which maintained the humidity close to zero. Because the water pressure gradient between the egg and the surroundings was constant, the water loss (measured by the drop in egg weight over several days) was proportional to the water conductance. Hence, the ratio of the average slope of the daily weight loss between intact and opened eggs represented the ratio of their respective conductances. In an additional set of EP embryos (N = 10), the measurements were performed as described above, except that the runs with the eggshell open (first at 38 °C, then at 30 °C) were performed in hyperoxia (about 35% O2), by delivering the hyperoxic gas inside the respirometer. Control measurements were performed to consider the possibility that the first cold-exposure, with the egg intact, may have some carry-over effects on the second thermogenic response, with the eggshell removed. To this end, measurements of the thermogenic responses were performed twice on control IP and EP eggs (N = 7 per group), following the same protocol of the experimental eggs, but without opening the eggshell. These eggs will be referred to as Controls. Cold-incubation These eggs were incubated at normal T (38 °C) until E18, at which time they were switched to 35 °C and left at this incubation T until they reached the EP phase (star fracture). Then, the thermogenic response was measured before and after the opening and removal of the eggshell, as described above. Quantification of thermogenesis, thermogenic response, and statistical analysis To statistically analyze. the thermogenic responses, first, for each animal, a plot of V O2 versus time was constructed beginning from the last 10 min at 38 °C until the end of the 1h exposure to 30 °C. The graph was printed on fixed coordinates, and the areas under the warm and the cold parts of the curve were digitized with a graphics tablet connected to a minicomputer. The area (ml). was divided by the time duration (min), yielding the average V O2 (ml/min) at 38 °C and at 30 °C; the ratio between the latter and the former gave the average magnitude of the thermogenic response to cold.
. All group data are presented as means ± 1 SEM. Values of V O2 in warm or cold conditions and the thermogenic responses at the various phases were compared among the five groups of animals (pre-IP, IP, EP, H1 b 8 h and H1 N 8 h) by ANOVA, followed by post-hoc Bonferroni's limitations for all possible comparisons. The effect of eggshell opening was evaluated as the ratio between the thermogenic response following and preceding eggshell removal by paired two-tailed t test. The same analysis was done for the comparison between the first and second run of the Control embryos. Finally, the percent changes caused by egg opening (relative to the intact condition) were compared to the percent differences between run 2 and run 1 of Controls by unpaired two-tailed t test. In all cases, significant differences were considered statistically significant at P b 0.05. Results Details about the number and body weight of the animals used for each particular experiment and age group are given in Table 1. Gaseous metabolism at 38 °C Fig. 1 provides . a visual . impression of the changes in gaseous metabolism (V O2 and V CO2) during the hatching process, from the IP phase until 12 .h after hatching. This representative recording shows that V O2 increased throughout the hatching period, with two major abrupt jumps, at the onset of the EP
Table 1 Number of animals, egg weight, age and body weight Age, days
N (N with yolk Egg weight Postincorporated) at E0, g hatching hours
1 — Development of the thermogenic response Pre-IP 19.7 ± 0.3 7 (0) 58.5 ± 2.4 IP 19.9 ± 0.2 17 (8) 58.1 ± 1.4 EP 19.9 ± 0.1 17 (16) 56.0 ± 1.1 H1 b 8 h 20.7 ± 0.3 a 7 (7) 57.1 ± 1.8 H1 N 8 h 20.0 ± 0.0 a 7 (7) 56.1 ± 0.8 E11 b 11 5 (0) 57.7 ± 1.2
– – – 3.1 ± 0.6 20.5 ± 1.1 –
2 — Thermogenic response before and after eggshell removal IP opening 20.3 ± 0.3 10 (6) 56.9 ± 2.0 – IP control 19.4 ± 0.2 7 (2) 59.7 ± 1.6 – EP opening 20.0 ± 0.0 10 (9) 56.3 ± 1.8 – EP control 20.0 ± 0.0 7 (7) 56.4 ± 1.0 –
Body weight, g
30.2 ± 2.2 36.0 ± 1.6 42.0 ± 0.8 40.6 ± 1.4 37.1 ± 0.8 4.0 ± 0.2
37.4 ± 1.9 35.6 ± 1.8 42.5 ± 1.2 40.5 ± 1.5
2 — Thermogenic response before and after eggshell removal, with hyperoxia EP 20.3 ± 0.2 10 (10) 61.3 ± 1.3 – 46.9 ± 0.9 3 — Thermogenic response before and after eggshell removal in cold-incubated embryos EP 20.4 ± 0.2 5 (5) 54.4 ± 2.6 – 40.2 ± 1.9 Values are mean ±1 SEM. N, number of animals. a In the hatchlings, age corresponds to the day of hatching. Pre-IP, IP, respectively before or after internal pipping. EP, during the star fracture of the external-pipping phase. H1 b 8 h, H1 b 8 h, hatchlings respectively during the first 8 h or the remaining 16 h of the first post-hatching day. b At E11, N refers to the number of sets, each set comprising two embryos.
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. . Fig. 1. Continuous measurements of oxygen consumption (VO2) and carbon dioxide production (VCO2) in one representative embryo from 12 h before to 12 h after hatching. The embryo was at the incubation temperature of 38 °C. EP indicates the onset of external pipping, with star fracture of the shell. Interruptions in the continuous lines indicate that the sampling was temporarily halted to check for baseline.
phase (star fracture) and at hatching itself. At this latter time, . V O2 approximately doubled, and gradually declined during the . following hours. The gradual increase in V O2 throughout the hatching phase was also apparent from the mean . values of the 5 groups measured at 38 °C. In fact, resting V O2 averaged about 0.3–0.35 ml/min in the pre-IP and IP embryos, 0.42 ml/min in the EP, and about 0.85 ml/min in the hatchlings (Fig. 2, left panel). The animal body weight (W) was similar among the preIP and IP embryonic stages; then, it increased in the EP embryos and hatchlings, because of the incorporation of the remaining yolk (Table 1).
Thermogenic responses at the various phases of the hatching process At all embryonic phases, exposure .to cold caused a very modest and short-lasting increase in V O2, followed by some decline (Fig. . 2, right panel). Hence, in the pre-IP, IP and EP embryos, V O2during the 1 h of cold averaged less than at 38 °C. Differently, in the hatchlings the thermogenic responses to cold were substantial, and the older hatchlings were able to maintain . V O2 better than the younger hatchlings. For comparison purposes this figure includes also the data of embryos at E11,
. Fig. 2. Left panel: oxygen consumption (VO2) measured continuously during the last 30 min at 38 °C, and one hour at . 30 °C, in embryos at different times of the hatching process, and in hatchlings. The vertical dashed line indicates the onset of the 30 °C exposure. Right panel: the VO2 data in the cold are plotted as percent of the 38 °C value preceding the onset of the cold (100%, horizontal dashed line). The fainter symbols refer to embryos studied at day 11 (E11), at which time they are strictly ectothermic, and the continuous line is the linear regression through the E11 data points. Symbols are mean values of 7 to 17 animals (or 5 sets of 2 embryos each in the case of E11). Bars indicate 1 SEM. Curves with different letters are statistically different from each other.
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passing of days. The average weight loss was 0.25 ± 0.01 g/day in the intact eggs, and 0.99 ± 0.04 g/day in those with eggshell opened (Fig. 3). Hence, opening the eggshell at its blunted end and removing about 1 cm2 increased gas-conductance by approximately 0.99/0.25 = 4 times. Thermogenesis after artificial opening the eggshell
Fig. 3. Weight of eggs maintained at a constant temperature in a desiccator for various days. Open symbols refer to intact eggs, filled symbols refer to eggs with the eggshell removed at its blunted end, for an area of about 1 cm2. The heavy and dashed lines are the linear regressions through the mean values of the intact and opened eggs, respectively.
i.e., at a developmental stage of pure ectothermy. All the curves were displayed above the E11 curve, indicating that also the preIP embryos presented some degree of endothermy, even though their thermogenic response was very modest. Opening the eggshell In the desiccator, at the constant water vapour pressure gradient of 21.3 mm Hg, eggs lost weight linearly with the
. At the IP stage, artificial opening of the eggshell raised V O2, especially during the cold-exposure (Fig. 4, top panel at right). On the other hand, simply repeating the cold exposure without opening the . shell (run 2 versus run 1 in Controls) did not increase V O2; rather, it had a small trend to lower it (Fig. 4, top panel at left). Therefore, the ratios of the thermogenic response with eggshell opened, relative to intact, were significantly higher than the ratios between run 2 and run 1 of Controls (P b 0.05). After normalisation by the values at 38 °C, the shape of the thermogenic curves appeared quite similar, both in Controls and in the experimental embryos (Fig. 4, bottom panels). In conclusion, in the embryos during the IP phase, opening the shell increased . the thermogenic capacity by raising the absolute values of V O2. Differently from the IP, at. the EP stage artificial opening of the eggshell did not alter V O2 at 38 °C, while it caused an important improvement of the thermogenic response (Fig. 5, top panel, at right) (P b 0.001). In Controls (Fig. 5, top panel at left) the differences between run 1 and 2 were not statistically significant. Therefore, in the cold, the ratios of the thermogenic responses with eggshell opened and eggshell intact were
. Fig. 4. Oxygen consumption (VO2) in warm conditions (38 °C) and during the 60 min of cold exposure (30 °C) in embryos during the internal pipping (IP) phase. At left are Controls (N = 7), run 1 and run 2 referring to the two consecutive thermogenic responses. At right are experimental embryos (N = 10), in the intact situation (open symbols), and with the eggshell opened (filled symbols). The bottom panels represent the thermogenic responses, where the values in the cold are expressed as percent of the warm values. The continuous oblique line represents the response of the purely ectothermic embryos, at day E11 (from Fig. 2). Symbols are group means, bars indicate 1 SEM.
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. Fig. 5. Oxygen consumption (VO2) in warm conditions (38 °C) and during the 60 min of cold exposure (30 °C) in embryos during the star fracture of the externalpipping (EP) phase. At left are Controls (N = 7), run 1 and run 2 referring to the two consecutive thermogenic responses. At right are represented the embryos of the experimental group, in the intact situation (open symbols), and with the eggshell removed from the blunted end of the egg (filled symbols). The bottom panels represent the thermogenic responses, where the values in the cold are expressed as percent of the warm values. The continuous oblique line represents the response of the purely ectothermic embryos, at day E11 (from Fig. 2). Symbols are group means, bars indicate 1 SEM.
significantly higher than the corresponding ratios between run 2 and run 1 of Controls (P b 0.05). After normalisation by the warm values, in Controls, the thermogenic response of run 2 differed significantly from that of run 1 (P b 0.01; Fig. 5, bottom panels). These differences were significantly smaller than those observed by opening the eggshell (top left and top . right panels; P b 0.05). In conclusions, not only the overall V O2, but also the thermogenic responses were significantly higher with the eggshell removed than in the intact condition. Eggshell opening and hyperoxia In EP embryos, artificial opening of the eggshell with ac. companying hyperoxia raised V O2 significantly both at 38 °C (P b 0.05) and during the cold exposure (P b 0.01; Fig. 6, top panel). Also the thermogenic response exceeded that measured in the intact eggs (P b 0.05; bottom panel). However, none of these changes differed significantly from those measured in the EP embryos by solely opening the eggshell. Eggshell opening in cold-incubated embryos The embryos that were maintained at 35 °C during the last three days of incubation went through the normal phases of hatching, although with a slight delay compared to those incubated at the normal 38 °C. Their thermogenic response to cold was tested at the time of the EP-star fracture, and it was very modest. In fact, during the last 10–15 min of the 1-h cold
. Fig. 6. Oxygen consumption (VO2) in warm conditions (38 °C) and during the 60 min of cold exposure (30 °C) in embryos during the star fracture of the external-pipping (EP) phase. Open symbols refer to the intact situation, filled symbols refer to the test with the eggshell removed at the blunted end of the egg and additional O2 (about 35% O2). Values are expressed in ml/min (top panel) and in percent of the values attained at 38 °C (bottom panel). Symbols are group means (N = 10), bars indicate 1 SEM.
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embryo being provided by the pregnant mother or by the incubating parent, while it becomes an essential means of thermoregulation after birth. Differently from the cardiorespiratory adjustments, little is known about the factors involved in the rapid development of thermogenesis during the perinatal period. In mammals, catecholamines and thyroid hormones (triiodothyronine) are involved in the maturation of thermogenesis (Power et al., 2004). In birds, the dependency on the thyroid function is supported by experiments showing that treatment with thiourea blocked the onset of thermogenesis (Tazawa et al., 1989; Freeman, 1971). With the current experiment we wanted to consider the role that oxygenation per se might have in the establishment of thermogenesis. The avian model offered the opportunity of exploring the role of oxygenation at precisely defined stages, because of its gradual transition between diffusive and convective gas exchange, and because it is free from the possibility that maternal or placental responses may interfere with the fetal development of thermoregulation. Methodological considerations
Fig. 7. Thermogenic response in embryos incubated at 35 °C during the last three days of incubation. All embryos were studied during the star fracture of the . external-pipping (EP) phase. At top, oxygen consumption (VO2) is expressed in absolute values (ml/min) during warm conditions (38 °C) and during the 1-hour . cold exposure (30 °C). At bottom, values of VO2 are expressed in percent of those at 38 °C. Open and filled symbols, refer, respectively, to the condition of intact eggshell and of eggshell removed at the blunted end of the egg. The continuous oblique line represents the response of the purely ectothermic embryos, at day E11 (from Fig. 2). Symbols indicate group means, bars indicate 1 SEM.
. exposure the percent change in V O2 of these embryos was similar to that of the ectothermic E11 embryos (Fig. 7, open symbols). Opening the eggshell produced no significant change in either thermogenesis or thermogenic response (Fig. 7, filled symbols). . For most of the experiments described above the changes in V CO2 with T, and the differences among groups or. conditions were qualitatively similar to those described for V O2. The EP embryos in hyperoxia represented the exception. In these em. bryos the increase in V in the cold was 21% less than the CO 2 . increase in V O2. Discussion In birds, as in mammals, the transition from prenatal to postnatal life involves abandoning the diffusive form of gas exchange (chorioallantoic or placental) to initiate gas convection (pulmonary ventilation). The success of this transition depends on specific respiratory and cardiovascular adjustments, including the closure of systemic shunts in favour of pulmonary circulation, a process that is under hormonal control but also depends on adequate oxygenation. Thermoregulation is another function drastically changed at birth. In fact, prenatally, endothermy is minimal, the heat requirements of the developing
The important rise in thermogenic capacity between the last phase of embryonic development and the first days after hatching had been reported frequently (Freeman, 1964; Dunn, 1976; Tazawa and Rahn, 1987; Nichelmann and Tzschentke, 2002; Mortola and Labbè, 2005; Dzialowski et al., 2007). However, to assess the role of oxygenation, first, we needed an accurate time-course of the development of thermogenesis and its response to cold throughout the hatching phases. We found, as expected, that also the pre-IP embryos have some degree of endothermy, as shown by a thermogenic level exceeding that of the ectothermic E11 embryos both in warm and cold conditions. However, the thermogenic response improved drastically in the transition from EP to early hatching (Fig. 2). Therefore we have chosen the IP and EP (star fracture) stages as the appropriate phases to examine the effect of oxygenation. Opening and removing the eggshell above the airspace, an intervention that caused some four-fold increase in O2conductance, was the method chosen to increase the embryo's oxygenation. We felt that this was the most appropriate way of mimicking the natural process of increased O2 availability at hatching. The alternate approach would have been to expose the embryos to hyperoxia, as recently done on emu embryos (Dzialowski et al., 2007). However, exposure of the whole chorioallantoic membrane to hyperoxia not only differs from the natural event but also risks diminishing its perfusion, which varies inversely with oxygenation (Strick et al., 1991; Burton and Palmer, 1992), hindering its gas exchange properties. In addition, the egg stores for O2, although probably much smaller than those for CO2 (Mortola and Besterman, 2007), can trap O2 during hyperoxia, giving the false impression of a high . embryonic V O2and low Respiratory Exchange Ratio, a situation . that jeopardizes the calculation of V O2 (Frappell et al., 1991). A potential problem was the fact that the IP and, especially, the EP phases last only a few hours. (see Fig. 1), and are characterized by a progressive rise in V O2 (Fig. 2). Indeed, the
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few-hour experimental periods needed to measure the effects of eggshell opening after the test with the eggshell intact must have included the natural progression of thermogenesis. For this reason we have compared the results of eggshell opening to those of control experiments on intact eggs, subjected to two consecutive runs that temporally matched those of the experimental protocol. Increased O2 conductance A 1-cm2 opening of the air cell may seem a trivial fraction of the total egg surface area of approximately 70 cm2. In reality, it represents a large increase (approximately 4-fold, Fig. 3) in eggshell conductance. Furthermore, opening the eggshell means that the O2 partial pressure (PO2) in the air cell rises by about 50%, from the end-incubation value of about 100 mm Hg (Wangensteen and Rahn, 1971) to 153 mm Hg (at 60% water vapour saturation). Høiby et al. (1983) commented that, even though only about one third of the total embryo's blood flow is exposed to the air cell, its complete O2-saturation would still represent a significant increase (about 16%) to the total O2 content of the arterialised blood returning to the embryo. This agrees with the PO2 values measured in the allantoic vessels of embryos developing in eggs with air cell opened (Nakazawa and Tazawa, 1988). The increase in oxygenation during the IP phase raised . resting V O2 both in warm and cold conditions, without changing their relative proportion. The result conforms to the idea . that, in the chicken embryo at term, O2 availability limits V O2 (Høiby et al., 1983; Burton and Tullett, 1985; Tazawa et al., 1992, also for review). The same has been noted in emu embryos during the . IP phase (Dzialowski et al., 2007). Indeed, the increase in V O2 that we observed by opening the airspace of the IP embryos averaged 12%, very close to the 13% reported by Høiby et al. (1983) with exposure to pure O2. Hence, during the IP phase, the increased metabolism at 38 °C and the increased thermogenesis in the cold after opening the shell can be explained by removal of the O2-limitation. Differently, in the EP embryos . at 38 °C, opening the eggshell did not cause any increase in V O2. At this time of the hatching process the star fracture probably resolved the O2 limitation of the embryo. at rest, as suggested also by the fact that the embryo's V O2 was higher than during the IP phase. On the other hand, the thermogenic response to cold drastically increased after removing the shell, indicating that the diffusion of O2 through the star fracture alone, although providing adequate oxygenation at 38 °C, was still representing a limitation in the cold, when O2 demands. are increased. The exposure to hyperoxia did not raise V O2 significantly more than already obtained by removing the eggshell. Furthermore, the magnitude of the increase in hyperoxia could be. overestimated, owing to the fact that some of the measured V O2 includes O2 physically dissolved in the yolk and other embryonic and extra-embryonic . fluids. Hence, in hyperoxia, changes in V CO2 are more representative of the changes in embryonic . metabolism, and they were smaller than the changes in V O2. In any case, our results showed that, by adding O2, there was no further
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improvement of the thermogenic response compared to the opening experiment without hyperoxia. This indicates that the opening of the eggshell as it occurs during the natural hatching process entirely removes the oxygen limitation, and is fully sufficient for the establishment of thermogenesis. In conclusion, by increasing the improving the embryo's O2 availability . eggshell O2 conductance raised V O2, relieving the O2 limitation that, in the EP phase, precluded the full manifestation of the thermogenic response. Opening the eggshell in cold-incubated embryos Cold-incubation during the last phases of incubation slows down growth and blunts the development of thermogenesis (Mortola, 2006). This was the case also of the cold-incubated EP embryos of this study. Hence, these growth-retarded embryos provided an opportunity to investigate the effect of oxygenation during the EP phase in embryos small for age, and with a more primitive thermogenic capacity. The results indicated that the oxygenation offered by eggshell opening did not improve their thermogenic response, contrary to what observed in normally incubated embryos (compare Figs. 5 and 7). Therefore, the increase in thermogenesis observed in normally incubated embryos by raising their O2 conductance is not a phenomenon time-linked to the hatching process; rather, it is linked to the level of maturity of thermogenesis. In other words, the stimulatory effects of oxygenation on thermogenesis do not depend on hatching and its associated events but on the developmental staging of thermogenesis itself. Therefore, if these data were extrapolated to the human case, one would anticipate that, during birth, oxygenation can improve heat production in infants at term, i.e., in those infants with thermoregulatory mechanisms in the process of maturation, but would be ineffectual on those premature infants who lack thermogenic abilities. Acknowledgement Funds for this research came from a Canadian Institute of Health Research grant. References Ar, A., Paganelli, C.V., Reeves, R.B., Greene, D.G., Rahn, H., 1974. The avian egg: water vapour conductance, shell thickness, and functional pore area. The Condor 76, 153–158. Black, J.L., Burggren, W.W., 2004. Acclimation to hypothermic incubation in developing chicken embryos (Gallus domesticus). I. Developmental effects and chronic and acute metabolic adjustments. Journal of Experimental Biology 207, 1543–1552. Burton, G.J., Palmer, M.E., 1992. Development of the chick chorioallantoic capillary plexus under normoxic and normobaric hypoxic and hyperoxic conditions: a morphometric study. Journal of Experimental Biology 262, 291–298. Burton, F.G., Tullett, S.G., 1985. Respiration of avian embryos. Comparative Biochemistry and Physiology A 82, 735–744. Dunn, E.H., 1976. Development of endothermy and existence energy expenditure of nestling double-crested cormorants. The Condor 78, 350–356. Dzialowski, E.M., Burggren, W.W., Komoro, T., Tazawa, H., 2007. Development of endothermic metabolic response in embryos and hatchlings of the
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